Arginine Conversion to Nitroxide by Tetrahydrobiopterin-free Neuronal Nitric-oxide Synthase
2000; Elsevier BV; Volume: 275; Issue: 43 Linguagem: Inglês
10.1074/jbc.m004337200
ISSN1083-351X
AutoresSubrata Adak, Qian Wang, Dennis J. Stuehr,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoWe studied catalysis by tetrahydrobiopterin (H4B)-free neuronal nitric-oxide synthase (nNOS) to understand how heme and H4B participate in nitric oxide (NO) synthesis. H4B-free nNOS catalyzed Arg oxidation toN ω-hydroxy-l-Arg (NOHA) and citrulline in both NADPH- and H2O2-driven reactions. Citrulline formation was time- and enzyme concentration-dependent but was uncoupled relative to NADPH oxidation, and generated nitrite and nitrate without forming NO. Similar results were observed when NOHA served as substrate. Steady-state and stopped-flow spectroscopy with the H4B-free enzyme revealed that a ferrous heme-NO complex built up after initiating catalysis in both NADPH- and H2O2-driven reactions, consistent with formation of nitroxyl as an immediate product. This differed from the H4B-replete enzyme, which formed a ferric heme-NO complex as an immediate product that could then release NO. We make the following conclusions. 1) H4B is not essential for Arg oxidation by nNOS, although it helps couple NADPH oxidation to product formation in both steps of NO synthesis. Thus, the NADPH- or H2O2-driven reactions form common heme-oxy species that can react with substrate in the presence or absence of H4B. 2) The sole essential role of H4B is to enable nNOS to generate NO instead of nitroxyl. On this basis we propose a new unified model for heme-dependent oxygen activation and H4B function in both steps of NO synthesis. We studied catalysis by tetrahydrobiopterin (H4B)-free neuronal nitric-oxide synthase (nNOS) to understand how heme and H4B participate in nitric oxide (NO) synthesis. H4B-free nNOS catalyzed Arg oxidation toN ω-hydroxy-l-Arg (NOHA) and citrulline in both NADPH- and H2O2-driven reactions. Citrulline formation was time- and enzyme concentration-dependent but was uncoupled relative to NADPH oxidation, and generated nitrite and nitrate without forming NO. Similar results were observed when NOHA served as substrate. Steady-state and stopped-flow spectroscopy with the H4B-free enzyme revealed that a ferrous heme-NO complex built up after initiating catalysis in both NADPH- and H2O2-driven reactions, consistent with formation of nitroxyl as an immediate product. This differed from the H4B-replete enzyme, which formed a ferric heme-NO complex as an immediate product that could then release NO. We make the following conclusions. 1) H4B is not essential for Arg oxidation by nNOS, although it helps couple NADPH oxidation to product formation in both steps of NO synthesis. Thus, the NADPH- or H2O2-driven reactions form common heme-oxy species that can react with substrate in the presence or absence of H4B. 2) The sole essential role of H4B is to enable nNOS to generate NO instead of nitroxyl. On this basis we propose a new unified model for heme-dependent oxygen activation and H4B function in both steps of NO synthesis. l-arginine calmodulin 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (6R)-5,6,7,8-tetrahydro-l-biopterin nitric-oxide synthase neuronal NO synthase oxygenase domain of nNOS calmodulin high performance liquid chromatography dithiothreitol N ω-hydroxy-l-Arg o-phthalaldehyde Nitric oxide (NO) is generated from l-arginine (Arg)1 by the NO synthases (NOSs). Three main isoforms of NOS are expressed in animals and differ regarding their primary sequence, tissue location, and means of regulation (1MacMicking J. Xie Q. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3501) Google Scholar, 2Craven S.E. Bredt D.S. Cell. 1998; 93: 495-498Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 3Michel T. Feron O. J. Clin. Invest. 1997; 100: 2417-2423Crossref PubMed Scopus (853) Google Scholar). All NOSs are homodimers with subunits comprising an N-terminal oxygenase domain with binding sites for heme, 6R-tetrahydrobiopterin (H4B), and Arg, and a C-terminal reductase domain with binding sites for FMN, FAD, and NADPH (4Marletta M.A. Hurshman A.R. Rusche K.M. Curr. Opin. Chem. Biol. 1998; 2: 656-663Crossref PubMed Scopus (203) Google Scholar, 5Stuehr D.J. Biochim. Biophys. Acta Bioenerg. 1999; 1411: 217-230Crossref PubMed Scopus (816) Google Scholar). A ∼20-amino acid consensus site for calmodulin (CaM) binding is located between the reductase and oxygenase domains of each NOS (6Ruan J. Xie Q. Hutchinson N. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The reductase domain transfers NADPH-derived electrons to the oxygenase domain in response to CaM binding, and this enables heme-dependent oxygen activation and stepwise conversion of Arg to NO and citrulline, withN-hydroxy-l-arginine (NOHA) being formed as an intermediate (4Marletta M.A. Hurshman A.R. Rusche K.M. Curr. Opin. Chem. Biol. 1998; 2: 656-663Crossref PubMed Scopus (203) Google Scholar, 5Stuehr D.J. Biochim. Biophys. Acta Bioenerg. 1999; 1411: 217-230Crossref PubMed Scopus (816) Google Scholar). Stoichiometry studies show that NOS consumes 1 mol of NADPH for Arg hydroxylation, and consumes an additional 0.5 mol of NADPH to oxidize NOHA to citrulline plus NO (7Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 8Abu-Soud H.M. Presta A. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10811-10816Crossref PubMed Scopus (72) Google Scholar). In the absence of Arg, NOS heme reduction leads to superoxide and H2O2 production (9Pou S. Keaton L. Surichamorn W. Rosen G.M. J. Biol. Chem. 1999; 274: 9573-9580Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 10Klatt P. Schmidt K. Uray G. Mayer B. J. Biol. Chem. 1993; 268: 14781-14787Abstract Full Text PDF PubMed Google Scholar). The NOS heme is ligated to a cysteine thiolate (11Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar, 12Fischmann T.O. Hruza A. DaNiu X. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Paul R. Lundell D.J. Narula S.K. Weber P.C. Nat. Struct. Biol. 1999; 6: 233-242Crossref PubMed Scopus (410) Google Scholar, 13Raman C.S. Li H. Martasek P. Kral V. Masters B.B.S. Poulos T.M. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar) and thus is likely to activate oxygen for substrate oxidation as occurs in the cytochrome P450 monooxygenases (14Mansuy D. Renaud J.P. Ortiz de Montellano P.R. Cytochrome P450 Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 537-574Crossref Google Scholar). In fact, both steps of NO synthesis are envisioned to involve heme-based oxygen activation and catalysis. However, NOS also requires H4B, and given that H4B is not required for heme reduction (15Presta A. Weber-Main A.M. Stankovich M.T. Stuehr D.J. J. Am. Chem. Soc. 1998; 120: 9460-9465Crossref Scopus (101) Google Scholar), it has been puzzling why this cofactor is essential for NO synthesis. H4B affects NOS in many ways; it stabilizes dimeric structure (16Klatt P. Schmidt K. Lehner D. Glatter O. Bachinger H.P. Mayer B. EMBO J. 1995; 14: 3687-3695Crossref PubMed Scopus (266) Google Scholar, 17Presta A. Siddhanta U. Wu C. Sennequier N. Huang L. Abu-Soud H.M. Erzurum S. Stuehr D.J. Biochemistry. 1998; 37: 298-310Crossref PubMed Scopus (150) Google Scholar), increases affinity for substrate (18McMillan K. Masters B.S.S. Biochemistry. 1993; 32: 9875-9880Crossref PubMed Scopus (164) Google Scholar, 19Klatt P. Schmid M. Leopold E. Schmidt K. Werner E.R. Mayer B. J. Biol. Chem. 1994; 269: 13861-13866Abstract Full Text PDF PubMed Google Scholar), speeds decay of the NOS ferrous-O2complex (20Abu-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 (159) Google Scholar), increases the rate of NADPH consumption (15Presta A. Weber-Main A.M. Stankovich M.T. Stuehr D.J. J. Am. Chem. Soc. 1998; 120: 9460-9465Crossref Scopus (101) Google Scholar, 21Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: 5566-5571Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and controls the midpoint potential of the heme (15Presta A. Weber-Main A.M. Stankovich M.T. Stuehr D.J. J. Am. Chem. Soc. 1998; 120: 9460-9465Crossref Scopus (101) Google Scholar). However, only H4B's ability to destabilize the ferrous-O2 complex appears linked to its essential role in NO synthesis. Aromatic amino acid hydroxylases use H4B as an electron donor for oxygen activation (22Kappock T.J. Caradonna J.P. Chem. Rev. 1996; 96: 1659-2756Crossref Scopus (290) Google Scholar, 23Kaufman S. Adv. Enzymol. Relat. Areas Mol. Biol. 1993; 67: 77-264PubMed Google Scholar), and a similar role for H4B in the NOS reaction has long been considered (24Bec N. Gorren A.C.F. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Recent crystal structures of NOS oxygenase domains (11Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar, 12Fischmann T.O. Hruza A. DaNiu X. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Paul R. Lundell D.J. Narula S.K. Weber P.C. Nat. Struct. Biol. 1999; 6: 233-242Crossref PubMed Scopus (410) Google Scholar, 13Raman C.S. Li H. Martasek P. Kral V. Masters B.B.S. Poulos T.M. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar) show that H4B binds near the heme edge and forms a hydrogen bond between N3 of its pterin ring and a heme propionate, consistent with a possible role in heme reduction. Indeed, Hurshman et al. (25Hurshman A.R. Krebs C. Edmondson D.E. Huynh B.H. Marletta M.A. Biochemistry. 1999; 38: 15689-15696Crossref PubMed Scopus (216) Google Scholar) recently used freeze-quench EPR to demonstrate that some H4B can convert to an H4B radical during reaction of ferrous NOS oxygenase domain with O2. Although this shows how H4B could participate in oxygen activation by donating an electron to the ferrous-dioxy heme, to what extent this takes place during normal NO synthesis is unknown. Clearly, electron donation from H4B is not required for NOHA oxidation, because H4B-free NOS catalyzes oxidation of NOHA to citrulline and nitroxyl (NO−) in either an NADPH- or H2O2-driven reaction (26Rusche K.M. Spiering M.M. Marletta M.A. Biochemistry. 1998; 37: 15503-15512Crossref PubMed Scopus (169) Google Scholar,27Clague M.J. Wishnok J.S. Marletta M.A. Biochemistry. 1997; 36: 14465-14473Crossref PubMed Scopus (75) Google Scholar). In these same systems, Arg oxidation was not observed, leading the authors to conclude that H4B is essential for Arg hydroxylation. However, this is a troublesome proposal because it means that H4B's role in oxygen activation depends on the identity of the substrate, or implies that reactivity of NOS heme-oxy species somehow differ in the presence or absence of H4B. In addition, it is still unclear how NOHA oxidation by H4B-bound NOS generates NO rather than NO−. To address these issues, we extensively investigated Arg and NOHA oxidation by H4B-free neuronal NOS (nNOS). Our results reveal that nNOS does indeed catalyze oxidation of both Arg 2While this manuscript was in review, Gacchui and colleagues (43Adhikari S. Ray S. Gachhui R. FEBS Lett. 2000; 475: 35-38Crossref PubMed Scopus (8) Google Scholar) reported that the H4B-free nNOS oxygenase domain converted Arg to citrulline and nitrite in an H2O2-supported reaction. and NOHA in the absence of H4B. This result, together with stopped-flow data, enable us to propose a simple, unified mechanism for heme-based oxygen activation, reactivity, and H4B participation in both steps of NO synthesis. H4B was purchased from Schirks Laboratory (Jona, Switzerland) and stock solutions prepared in 3 mdithiothreitol (DTT). 2′,5′-ADP Sepharose 4B was purchased from Alexis Corp. NOHA was a gift from Dr. Bruce King (Wake Forest University, Winston-Salem, NC). All other reagents and materials were obtained from Sigma or from sources reported previously (28Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Full-length nNOS containing a six-histidine tag at its N terminus was overexpressed inEscherichia coli using the PCWori vector and purified as reported previously (28Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The protein as isolated was free of Arg and H4B and was low spin as judged by a Soret peak at 420 nm. The nNOS concentration was estimated based on the absorbance of its ferrous heme-CO adduct as previously reported (28Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The nNOS oxygenase domain (nNOSoxy; amino acids 1–720) containing a six-histidine tag at its C terminus was also expressed in E. coli and purified in the absence of H4B as described previously (29Huang L. Abu-Soud H.M. Hille R. Stuehr D.J. Biochemistry. 1999; 38: 1912-1920Crossref PubMed Scopus (47) Google Scholar). The initial rate of NO synthesis by nNOS was quantitated at 25 °C using the oxyhemoglobin assay for NO (28Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). nNOS was added to a cuvette containing 40 mm EPPS, pH 7.6, 150 μg/ml CaM, 0.62 mmCaCl2, 10 units/ml superoxide dismutase, 0.3 mmDTT, 5 mm Arg, 4 μm each of FAD and FMN, 100 units/ml catalase, and 10 μm oxyhemoglobin. NO-mediated conversion of oxyhemoglobin to methemoglobin was monitored over time at 401 nm and quantitated using a difference extinction coefficient of 38,000 m−1cm−1. NADPH oxidation at 25 °C was quantitated at 340 nm using an extinction coefficient of 6,220m−1 cm−1. The assay mixture was identical to the NO synthesis measurement except that oxyhemoglobin was absent. H2O2-dependent nNOS oxidation of Arg or NOHA to nitrite was assayed in 96-well microplates at 25 °C. Each well contained 40 mm EPPS, pH 7.6, 0.6 μm nNOS, 1 mm Arg, 1 mmDTT, 25 units/ml superoxide dismutase, 0.5 mm EDTA, and with or without 100 μm H4B. For NOHA oxidation the enzyme concentration was reduced to 0.06 μm. Reactions were initiated by adding 30 mm H2O2 and stopped after 10 min by adding 1300 units of catalase. Nitrite was detected at 550 nm using the Griess reagent (100 μl) and quantitated based on a nitrite standard curve. H4B-free nNOS (0.5 μm) or H4B-bound nNOS (50 nm) was added to 96-well microplates containing 40 mm EPPS, pH 7.6, 150 μg/ml CaM, 0.62 mmCaCl2, 0.3 mm DTT, 1 mm Arg or NOHA, 4 μm each of FAD and FMN, 100 units catalase, and 25 units/ml superoxide dismutase to give a final volume 100 μl. The reaction was started by adding NADPH to give 1.0 mm and stopped after 25 min by adding excess EDTA. Nitrate reductase (1 μm) was added, and the plates were incubated for 2 h at room temperature to reduce nitrate. Excess NADPH was then oxidized by adding lactate dehydrogenase and pyruvate, and total nitrite plus nitrate was detected by adding Griess reagent and reading at 550 nm. Optical spectra were recorded on a Hitachi 3010 UV-visible spectrophotometer. Anaerobic spectra were recorded using septum-sealed quartz cuvettes that could be attached through a quick-fit joint to a vacuum system. The nNOS samples were made anaerobic by repeated cycles of evacuation and equilibrated with catalyst-deoxygenated N2. A separate buffer solution containing cofactors was evacuated and gassed with N2 in a separate vessel and then transferred into the anaerobic cuvette. All transfers were made using gas tight syringes. The stopped-flow instrument was attached to a rapid-scanning diode array device (Hi-Tech model MG-6000) designed to collect 96 spectra in a specific time frame. Experiments for H2O2-dependent reactions involved mixing an anaerobic solution containing 6 μm nNOSoxy and 1 mm NOHA with anaerobic buffer solution containing 2 mm H2O2. In some cases the enzyme solution also contained 10 μm H4B. Experiments with O2-dependent reactions involved mixing an anaerobic solution of 16 μm ferrous nNOSoxy containing 20 μm H4B and 2 mm NOHA with aerobic buffer solution. H4B-free nNOS was diluted to 4 μm in air-saturated 40 mm EPPS buffer, pH 7.6, containing 6.0 μm CaM, 0.2 mm DTT or β-mercaptoethanol, 0.9 mm EDTA, 24 μm NADPH, 1.0 mmArg or NOHA, 0.5 mm glucose 6-phosphate, and 2 units of glucose-6-phosphate dehydrogenase; final volume was 0.5 ml. Reactions were started by adding 2 mm Ca2+ and monitored by wavelength scanning at 25 °C in the Hitachi U3010 spectrophotometer. In some cases, substrate was omitted and CO gas was bubbled into the cuvette after initiating catalysis as explained in the text. For stopped-flow rapid scanning experiments, conditions were identical except that an NADPH-regenerating system was not used; the final concentrations of NADPH and nNOS were 40 and 2 μm, respectively, and the temperature was 15 °C. Amino acids were derivatized by o-phthalaldehyde (OPA) and separated by reverse-phase HPLC, using an Waters model 510 instrument with a Waters 470 scanning fluorescence detector, Waters 715 Ultra Wisp Sampler Processor, and a Hewlett Packard ODS Hypersil 5-μm 100 × 21-mm C18 column (Hewlett Packard 79916 OD-552), equipped with a C18 guard column (2.1 mm, inner diameter, ODS Hypersil, 5 μm, Hewlett Packard 79916KT-110). The injector was set to mix 60 μl of an OPA reaction solution (4 mg of OPA dissolved in 0.5 ml of methanol to which 4.5 ml of 0.1 m sodium borate, pH 10, and 30 μl of β-mercaptoethanol were added) with a 40-μl reaction sample. After reacting for 2 min, the samples were automatically applied to the column, which was equilibrated with 5 mm ammonium acetate (pH 6.0) containing 20% methanol (v/v) (solvent A) run at 0.5 ml/min at room temperature. The elution conditions for the OPA derivatives were 0–50% solvent B (methanol) over 9 min, followed by a linear increase to 100% methanol over the next 0.5 min, 100% methanol for 3 min, and a return to 100% solvent A over the next 0.5 min. Amino acid standards were used to quantify the samples. Citrulline, NOHA, and Arg standards had retention times of.6.2, 10.2, and 12.1 min, respectively, and the peaks were completely resolved. Amino acids were detected by fluorescence emission (excitation 360 nm and emission 455 nm) and quantitated based on authentic prepared standards. H4B-free nNOS generated detectable amounts of citrulline 3Under the conditions of our HPLC analysis, any cyanoornithine produced would have been detected as citrulline. and nitrite plus nitrate from Arg when the reactions contained relatively high concentrations of enzyme (Fig. 1, TableI). NOHA was also detected as a product, indicating that it forms as an intermediate in the reaction catalyzed by H4B-free enzyme. When NOHA was used in place of Arg as a substrate it also was converted to citrulline and nitrite plus nitrate by H4B-free nNOS (Fig. 1, Table I), as shown previously with inducible NOS (26Rusche K.M. Spiering M.M. Marletta M.A. Biochemistry. 1998; 37: 15503-15512Crossref PubMed Scopus (169) Google Scholar). Citrulline formation from Arg or NOHA was 10 or 15%, respectively, compared with the H4B-replete enzyme 4The activity of our H4B-reconstituted nNOS was 25 ± 2 min−1 at room temperature when assayed by the oxyhemoglobin assay. This represents 50% of the activity we observe for nNOS purified in the presence of H4B. in reactions run under otherwise identical conditions (Table I). A somewhat greater proportion of nitrate was generated in reactions catalyzed by the H4B-free enzyme. Control reactions run in the absence of substrate, nNOS, or NADPH did not generate detectable citrulline or nitrite plus nitrate. Including catalase and superoxide dismutase did not diminish product formation in any case. We conclude that H4B is not essential for nNOS to oxidize either Arg or NOHA to citrulline and nitrite plus nitrate in the NADPH-driven reaction. Our results are the first to show that the Arg reaction can occur in an H4B-free NOS.2 As shown in Fig. 2, NADPH-driven citrulline formation from Arg or NOHA was time-dependent (left panel) and gave initial rates of 2 and 5 min−1, respectively. The reaction was also dependent on nNOS concentration until it reached a point where all NADPH was exhausted within the time of assay (above 500 nmnNOS, right panel). Importantly, citrulline production was associated with no detectable NO synthesis, even under assay conditions where a single NO per heme would have been detected. This implies the H4B-free nNOS formed a nitrogen oxide product other than NO that could oxidize to nitrite and nitrate.Table IComparative activities of nNOS in NADPH-driven reactions at 25 °CExperimentCitrulline producedNOHA producedNO2− + NO3− producedmol/mol nNOSmol/mol nNOSmol/mol nNOSE + Arg + NADPH 1-aE, nNOS; NA, not applicable; ND, not detectable.44 ± 428 ± 245 ± 5E + Arg + H4B + NADPH458 ± 3082 ± 8352 ± 30E + NOHA + NADPH108 ± 10NA70 ± 6E + NOHA + H4B + NADPH745 ± 60NA554 ± 50Arg + H4B + NADPHNDNDNDE + H4B + NADPHNDNDNDE + H4B + ArgNDNDNDIncubations were run for 25 min prior to quenching as described under "Experimental Procedures." The values are the mean ± S.D. of three measurements.1-a E, nNOS; NA, not applicable; ND, not detectable. Open table in a new tab Figure 2Time- and concentration-dependent activities of H4B-free nNOS. Panel A depicts citrulline accumulation during NOHA or Arg oxidation. Panel B shows rates of citrulline formation and NO synthesis from Arg at different concentrations of enzyme. Assay conditions were identical to Fig. 1 except these reactions were started by adding 0.2 mm NADPH and were terminated after 5 min by adding 0.6n hydrochloric acid. Citrulline was measured by a fluorometric HPLC method, and NO synthesis was measured by the oxyhemoglobin assay. Each point is the mean of three measurements.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Incubations were run for 25 min prior to quenching as described under "Experimental Procedures." The values are the mean ± S.D. of three measurements. We next determined the stoichiometric relationship between NADPH consumption and citrulline formation by H4B-free nNOS (Fig.3). Reactions contained different amounts of NADPH, and the total citrulline produced was determined after all the NADPH was consumed. The slopes indicate that conversion of 1 mol of Arg or NOHA to citrulline was associated with oxidation of 16 and 6 mol of NADPH, respectively. For the same nNOS preparation made replete with H4B, the values were approximately 2 NADPH oxidized per citrulline from Arg, and 0.5 NADPH oxidized per citrulline from NOHA (data not shown), which are close to the theoretical minimum values (7Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). The uncoupling seen under H4B-free conditions was not due to structural changes, because H2B, which mimics all the structural effects of H4B (17Presta A. Siddhanta U. Wu C. Sennequier N. Huang L. Abu-Soud H.M. Erzurum S. Stuehr D.J. Biochemistry. 1998; 37: 298-310Crossref PubMed Scopus (150) Google Scholar, 30Crane B.R. Arvai A.S. Ghosh S. Getzoff E.D. Stuehr D.J. Tainer J.A. Biochemistry. 2000; 39: 4608-4621Crossref PubMed Scopus (143) Google Scholar), did not enhance coupling (data not shown). Our analysis indicates that NADPH oxidation in H4B-free nNOS is uncoupled from either Arg or NOHA oxidation to citrulline in a multiple turnover setting. To investigate the mechanism of the H4B-independent reaction and identify the nitrogen oxide product, we utilized spectroscopy to observe the enzyme during catalysis. In Fig. 4spectra of H4B-free nNOS were recorded before or after initiating its aerobic NADPH oxidation at 25 °C in the absence or presence of substrate. Sequential scans were recorded of the CaM-free resting ferric enzyme, after adding NADPH to reduce the flavins, and after adding Ca2+ to trigger CaM binding, heme reduction, and catalysis. In the absence of substrate (panel A), the light absorbance spectrum of the CaM- and H4B-free ferric NOS was a mixture of high and low spin heme with a prominent heme Soret band at 420 nm, consistent with its lack of bound H4B (31Roman L.J. Sheta E.A. Martasek P. Gross S.S. Lin Q. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar). Adding NADPH caused losses in visible absorbance between 360 and 420 nm, 440 and 520 nm, and 560 and 680 nm, and buildup of a broad absorbance centered near 365 nm, consistent with reduction of nNOS flavins. After adding Ca2+ to trigger steady state NADPH oxidation, we observed some decrease in absorbance between 390 and 420 nm and at 650 nm, consistent with heme reduction occurring in the substrate- and H4B-free nNOS (15Presta A. Weber-Main A.M. Stankovich M.T. Stuehr D.J. J. Am. Chem. Soc. 1998; 120: 9460-9465Crossref Scopus (101) Google Scholar). The inset of panel A also shows significant heme reduction occurred as is evidenced by buildup of a 444-nm ferrous-CO species after the reaction was given CO gas. 5When the H4B- and Arg-free enzyme was reduced with dithionite, 95% of its heme formed a 444-nm complex with CO (data not shown). The residual peak at 420 nm in the inset to Fig.4 therefore represents ferric low spin nNOS. This likely is the monomer that is present when nNOS is purified in the absence of Arg and H4B, whose heme cannot accept NADPH-derived electrons. As expected, the spectrum taken of the substrate- and H4B-free enzyme during steady state NADPH oxidation showed no evidence for buildup of a six-coordinate ferric or ferrous heme-NO complex, which display Soret absorbance bands at 436 and 440 nm, respectively (32Wang J. Rousseau D.L. Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10512-10516Crossref PubMed Scopus (126) Google Scholar). In the presence of Arg or NOHA (Fig. 4, panels B andC), the heme Soret band of the initial H4B-free ferric nNOS was broader with maximum at 398 nm, consistent with substrate binding and greater high spin character (31Roman L.J. Sheta E.A. Martasek P. Gross S.S. Lin Q. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar). The spectra recorded just after initiating steady-state oxidation of Arg or NOHA showed a small buildup of heme-NO complex in both cases (data not shown). The proportion of this species grew over time as the O2 was consumed in the cuvette. Spectra taken at 10 min (Fig. 4, panels B and C) clearly show the presence of six-coordinate ferrous-NO complex in the Arg and NOHA reactions, as evidenced by the shoulder near 436 nm and single broad absorbance peak near 570 nm (32Wang J. Rousseau D.L. Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10512-10516Crossref PubMed Scopus (126) Google Scholar). The nature of the heme-NO species is further defined by the difference spectra in panels B andC, which show absorbance maxima at 436 and 570 nm. Together, our results suggest that the immediate inorganic product of Arg or NOHA oxidation is nitroxide (NO−), which binds to the ferric heme to form a ferrous heme-NO complex. We next examined the kinetics of ferrous-NO complex formation in the H4B-free nNOS and its effect on the NADPH oxidation rate. Theleft panels of Fig.5 depict ferrous-NO complex formation during the initial phase of Arg or NOHA oxidation at 15 °C. The reactions were started by rapid mixing a solution of Ca2+with a solution containing CaM, nNOS, substrate, EDTA, and excess NADPH. In both cases, the absorbance increase at 436 nm was best fit to a two-exponential equation, giving apparent rate constants that are listed in Table II. These values indicate that heme-NO complex formation was biphasic and had the same kinetics whether Arg or NOHA serve as substrate in the H4B-free enzyme. The rates obtained for the H4B-free nNOS are similar to the kinetics of heme-NO complex buildup in H4B-saturated nNOS (33Abu-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 (201) Google Scholar, 34Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 17434-17439Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), but differ in two ways. First is the magnitude of absorbance gain at 436, which when normalized on a per heme basis indicate that the proportion of enzyme that forms the heme-NO complex during Arg or NOHA oxidation is small for H4B-free nNOS (approximately 10%) compared with H4B-saturated nNOS (approximately 70%; Ref. 33Abu-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 (201) Google Scholar). Second, the relative absorbance change due to the fast phase of complex buildup in the H4B-free enzyme differs from the H4B-saturated enzyme (33Abu-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 (201) Google Scholar, 34Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 17434-17439Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). As shown in theright panels of Fig. 5, rates of NADPH oxidation were not slowed by ferrous-NO complex buildup in H4B free nNOS, consistent with the small proportion of complex that is obs
Referência(s)