Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase
2008; Elsevier BV; Volume: 283; Issue: 17 Linguagem: Inglês
10.1074/jbc.m709250200
ISSN1083-351X
AutoresChin‐Chuan Wei, Zhiqiang Wang, Jesús Tejero, Ya‐Ping Yang, Craig Hemann, Russ Hille, Dennis J. Stuehr,
Tópico(s)Hemoglobin structure and function
ResumoNitric-oxide synthases (NOS) are catalytically self-sufficient flavo-heme enzymes that generate NO from arginine (Arg) and display a novel utilization of their tetrahydrobiopterin (H4B) cofactor. During Arg hydroxylation, H4B acts as a one-electron donor and is then presumed to redox cycle (i.e. be reduced back to H4B) within NOS before further catalysis can proceed. Whereas H4B radical formation is well characterized, the subsequent presumed radical reduction has not been demonstrated, and its potential mechanisms are unknown. We investigated radical reduction during a single turnover Arg hydroxylation reaction catalyzed by neuronal NOS to document the process, determine its kinetics, and test for involvement of the NOS flavoprotein domain. We utilized a freeze-quench instrument, the biopterin analog 5-methyl-H4B, and a method that could separately quantify the flavin and pterin radicals that formed in NOS during the reaction. Our results establish that the NOS flavoprotein domain catalyzes reduction of the biopterin radical following Arg hydroxylation. The reduction is calmodulin-dependent and occurs at a rate that is similar to heme reduction and fast enough to explain H4B redox cycling in NOS. These results, in light of existing NOS crystal structures, suggest a "through-heme" mechanism may operate for H4B radical reduction in NOS. Nitric-oxide synthases (NOS) are catalytically self-sufficient flavo-heme enzymes that generate NO from arginine (Arg) and display a novel utilization of their tetrahydrobiopterin (H4B) cofactor. During Arg hydroxylation, H4B acts as a one-electron donor and is then presumed to redox cycle (i.e. be reduced back to H4B) within NOS before further catalysis can proceed. Whereas H4B radical formation is well characterized, the subsequent presumed radical reduction has not been demonstrated, and its potential mechanisms are unknown. We investigated radical reduction during a single turnover Arg hydroxylation reaction catalyzed by neuronal NOS to document the process, determine its kinetics, and test for involvement of the NOS flavoprotein domain. We utilized a freeze-quench instrument, the biopterin analog 5-methyl-H4B, and a method that could separately quantify the flavin and pterin radicals that formed in NOS during the reaction. Our results establish that the NOS flavoprotein domain catalyzes reduction of the biopterin radical following Arg hydroxylation. The reduction is calmodulin-dependent and occurs at a rate that is similar to heme reduction and fast enough to explain H4B redox cycling in NOS. These results, in light of existing NOS crystal structures, suggest a "through-heme" mechanism may operate for H4B radical reduction in NOS. Nitric-oxide synthases (NOS, 3The abbreviations used are: NOS, nitric-oxide synthase; NOSoxy, NOS oxygenase domain; NOSred, NOS reductase domain; NOHA, Nω-hydroxyl-l-arginine; H4B, (6R)-5,6,7,8-tetrahydro-l-biopterin; 5MeH4B, (6R)-5-methyl-6,7,8-trihydro-l-biopterin; FeIII (FeIIIO2−), heme ferric superoxy intermediate; EPPS, N-(2-hydroxyethyl)-piperazine-N′-3-propanesulfonic acid; FSQ, flavin semiquinone; EPR, electron paramagnetic resonance; CaM, calmodulin; nNOS, neuronal NOS. 3The abbreviations used are: NOS, nitric-oxide synthase; NOSoxy, NOS oxygenase domain; NOSred, NOS reductase domain; NOHA, Nω-hydroxyl-l-arginine; H4B, (6R)-5,6,7,8-tetrahydro-l-biopterin; 5MeH4B, (6R)-5-methyl-6,7,8-trihydro-l-biopterin; FeIII (FeIIIO2−), heme ferric superoxy intermediate; EPPS, N-(2-hydroxyethyl)-piperazine-N′-3-propanesulfonic acid; FSQ, flavin semiquinone; EPR, electron paramagnetic resonance; CaM, calmodulin; nNOS, neuronal NOS. EC 1.14.13.39) are flavo-heme enzymes that catalyze a stepwise oxidation of l-arginine (Arg) to form nitric oxide (NO) and l-citrulline (1Gorren A.C.F. Mayer B. Biochim. Biophys. Acta. 2007; 1770: 432-445Crossref PubMed Scopus (107) Google Scholar, 2Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (165) Google Scholar) (Fig. 1). Arg is first hydroxylated by NOS in an NADPH- and O2-dependent reaction to form Nω-hydroxyl-l-arginine (NOHA) as an enzyme-bound intermediate. NOHA is then oxidized by NOS to citrulline and NO in a second NADPH- and O2-dependent reaction. The reactions take place within the NOS oxygenase domain dimer (NOSoxy), which contains bound protoporphyrin IX (heme) and 6-(R)-tetrahydrobiopterin (H4B). The NOS heme catalyzes oxygen activation by a mechanism that is similar to that described for cytochrome P450 (3Denisov I.G. Makris T.M. Sligar S.G. Schlichting I. Chem. Rev. 2005; 105: 2253-2277Crossref PubMed Scopus (1525) Google Scholar). However, in NOS the electron required for ferric heme reduction is provided by an attached flavoprotein domain that contains FAD, FMN, and an NADPH binding site, and heme reduction is dependent on calmodulin (CaM) binding (1Gorren A.C.F. Mayer B. Biochim. Biophys. Acta. 2007; 1770: 432-445Crossref PubMed Scopus (107) Google Scholar, 2Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (165) Google Scholar). NOS enzymes also require H4B as a cofactor. H4B is bound tightly within NOS enzymes (koff ranges from 0.07 to 1.6 min-1 at 37 °C) (4List 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, 5Gorren A.C.F. List B.M. Schrammel A. Pitters E. Hemmens B. Werner E.R. Schmidt K. Mayer B. Biochemistry. 1996; 35: 16735-16745Crossref PubMed Scopus (142) Google Scholar), and this enables H4B to remain bound in NOS through multiple catalytic turnovers (6Reif A. Frohlich L.G. Kotsonis P. Frey A. Bommel H.M. Wink D.A. Pfleiderer W. Schmidt H.H. J. Biol. Chem. 1999; 274: 24921-24929Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 7Giovanelli J. Campos K.L. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7091-7095Crossref PubMed Scopus (148) Google Scholar, 8Witteveen C.F.B. Giovanelli J. Kaufman S. J. Biol. Chem. 1999; 274: 29755-29762Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Moreover, H4B performs a novel redox function in NOS: it reduces the ferric heme-superoxy intermediate (FeIII (FeIIIO2−)) that forms during oxygen activation, and becomes an enzyme-bound H4B radical in the process (9Hurshman A.R. Krebs C. Edmondson D.E. Marletta M.A. Biochemistry. 2003; 42: 13287-13303Crossref PubMed Scopus (51) Google Scholar, 10Berka V. Yeh H.C. Gao D. Kiran F. Tsai A.L. Biochemistry. 2004; 43: 13137-13148Crossref PubMed Scopus (46) Google Scholar, 11Schmidt P.P. Lange R. Gorren A.C.F. Werner E.R. Mayer B. Andersson K.K. J. Biol. Inorg. Chem. 2001; 6: 151-158Crossref PubMed Scopus (94) Google Scholar, 12Wei C.C. Wang Z.Q. Durra D. Hemann C. Hille R. Garcin E.D. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 2005; 280: 8929-8935Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13Wei 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). Electron transfer from H4B is critical because it enables NOS to form the heme-oxy species that react with Arg or NOHA (1Gorren A.C.F. Mayer B. Biochim. Biophys. Acta. 2007; 1770: 432-445Crossref PubMed Scopus (107) Google Scholar, 2Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (165) Google Scholar) (Fig. 1). Several studies suggest that the H4B radical remains in NOS after it forms and must be reduced back to H4B in order for the enzyme to continue catalysis (1Gorren A.C.F. Mayer B. Biochim. Biophys. Acta. 2007; 1770: 432-445Crossref PubMed Scopus (107) Google Scholar, 2Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (165) Google Scholar) (Fig. 1). However, the fact that bound H4B radical undergoes a time-dependent oxidation (10Berka V. Yeh H.C. Gao D. Kiran F. Tsai A.L. Biochemistry. 2004; 43: 13137-13148Crossref PubMed Scopus (46) Google Scholar, 12Wei C.C. Wang Z.Q. Durra D. Hemann C. Hille R. Garcin E.D. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 2005; 280: 8929-8935Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13Wei 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), and that NOHA can dissociate from NOS after it forms (14Stuehr D.J. Kwon N.S. Nathan C.F. Griffith O.W. Feldman P.L. Wiseman J. J. Biol. Chem. 1991; 266: 6259-6263Abstract Full Text PDF PubMed Google Scholar, 15Buga G.M. Singh R. Pervin S. Rogers N.E. Schmitz D.A. Jenkinson C.P. Cederbaum S.D. Ignarro L.J. Am. J. Physiol. 1996; 40: H1988-H1998Google Scholar), places a time constraint on H4B radical reduction. Following Arg hydroxylation, the H4B radical remains in the ferric enzyme (Fig. 1, enzyme species III), and thus its reduction should require an electron from the NOS flavoprotein domain (1Gorren A.C.F. Mayer B. Biochim. Biophys. Acta. 2007; 1770: 432-445Crossref PubMed Scopus (107) Google Scholar, 2Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (165) Google Scholar) (Fig. 1, enzyme species III and IV). The validity of this process and its regulation are unclear. Because CaM binding to NOS enables the flavoprotein domain to transfer electrons to the oxygenase domain heme (16Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar), we hypothesized that reduction of the H4B radical following Arg hydroxylation may also require CaM binding to NOS. To explore this possibility, we developed a means to monitor the extent and kinetics of pterin radical reduction in NOS after Arg hydroxylation occurs. Our experiments manipulate free Ca2+ levels so as to allow or prevent the CaM-dependent electron transfer between the NOS flavoprotein and oxygenase domains, as we have done previously (17Abu-Soud H.M. Presta A. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10811-10816Crossref PubMed Scopus (72) Google Scholar). We also utilize an EPR technique that can distinguish the radical signals arising from the bound pterin versus the NOS flavins during the reaction. Our results provide the first example of enzyme-catalyzed pterin radical reduction in biology, and lay a foundation to explore its mechanism and regulation in the NOS enzymes. Chemicals—H4B, H2B, and 5-methyl-H4B (5MeH4B) were obtained from Dr. Schirck's laboratory (Jona, Switzerland). A 5MeH4B stock solution was prepared fresh in 40 mm EPPS, pH 7.5. All other chemicals were obtained either from Sigma or Fisher Scientific International, Inc. UV-visible Spectrometry—Conventional spectra were obtained using a Cary 100 BIO instrument (Varian, Inc) equipped with temperature control and automatic stirring. Protein Purification—Rat nNOS was overexpressed in Escherichia coli and was purified in the absence of H4B as described previously (18Adak 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, 19Panda K. Adak S. Aulak K.S. Santolini J. McDonald J.F. Stuehr D.J. J. Biol. Chem. 2003; 278: 37122-37131Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The enzyme concentration was determined from the 444-nm absorbance of the ferrous-CO complex using an extinction coefficient of 76 mm-1 cm-1. The individual nNOSoxy and flavoprotein domains were overexpressed and purified as described previously (19Panda K. Adak S. Aulak K.S. Santolini J. McDonald J.F. Stuehr D.J. J. Biol. Chem. 2003; 278: 37122-37131Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 20Konas D.W. Takaya N. Sharma M. Stuehr D.J. Biochemistry. 2006; 45: 12596-12609Crossref PubMed Scopus (20) Google Scholar). The concentrated proteins were stored in a buffer containing 50 mm EPPS, pH 7.5, 2 mm β-mercaptoethanol, 10% glycerol, and 0.25 m NaCl. Protein Sample Preparation—Ferrous nNOS was prepared by reducing CaM-bound ferric enzyme with NADPH in a gastight cuvette under anaerobic conditions as described previously (19Panda K. Adak S. Aulak K.S. Santolini J. McDonald J.F. Stuehr D.J. J. Biol. Chem. 2003; 278: 37122-37131Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 20Konas D.W. Takaya N. Sharma M. Stuehr D.J. Biochemistry. 2006; 45: 12596-12609Crossref PubMed Scopus (20) Google Scholar). Briefly, a buffered solution containing ∼120 μm nNOS, 10 mm Arg, 2 mm 5MeH4B (or 1 mm H2B), 200 μm CaM, and 600 μm Ca2+ was made anaerobic by several cycles of vacuum and flushing with deoxygenated N2, and then had N2-purged NADPH solution added to give 0.1 to 0.2 mm NADPH. Heme reduction was monitored by the appearance of a 550-nm absorbance peak and the disappearance of the 650-nm absorbance peak. After heme reduction was judged to be complete, we added additional NADPH to give a concentration of ∼1 mm. In some cases, an anaerobic EDTA solution was then added to the ferrous NOS sample to give 1.2 mm EDTA to release the bound CaM from the ferrous nNOS. The samples were periodically scanned in the UV-visible spectrophotometer to assure that the ferrous heme did not oxidize to ferric heme prior to transfer to the rapid quench instrument. Rapid-freeze Kinetic Experiments—Ferrous nNOS samples prepared as described above were transferred with an anaerobic syringe to a rapid quench instrument (RQF-63, TGK Scientific, Bradford on Avon, UK) maintained at 10 °C, and the samples were rapid-mixed with an O2-saturated buffer (40 mm EPPS, 125 mm NaCl, pH 7.5) to initiate the reaction (this resulted in a post-mix O2 concentration in the reaction of ∼0.8 mm). The reaction mixture was then aged for various times in the instrument followed by rapid injection into a liquid N2/isopentane freezing solution as described elsewhere (21Wei 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). Some replicate reactions utilized H2B-bound nNOS in the rapid-freeze EPR study, to exclusively monitor the flavin semiquinone (FSQ) radical during the single turnover reaction. (H2B does not form a radical in NOS during catalysis; Refs. 13Wei 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, 21Wei 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.) Three different nNOS preparations were used in the experiments, and each nNOS preparation was reacted under the CaM-bound and CaM-free condition on the same day. EPR Analysis—Electron paramagnetic resonance (EPR) spectra of each frozen reaction sample was recorded in a Bruker ER300 instrument equipped with an ER 035 NMR gauss meter and a Hewlett-Packard 5352B microwave power controller. Temperature control was achieved using Oxford Instruments ESR 900 continuous-flow liquid helium cryostat and ITC4 temperature controller. All spectra were recorded at 150 K using a microwave power of 2 milliwatts or 20 microwatts, a frequency of 9.5 GHz, a modulation amplitude of 5 G, and a modulation frequency of 100 kHz. The EPR spectrum of the 5MeH4B radical was obtained by mixing 5MeH4B-bound ferrous nNOSoxy with O2-saturated buffer and then rapid freezing after 125 ms of reaction as performed previously (12Wei C.C. Wang Z.Q. Durra D. Hemann C. Hille R. Garcin E.D. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 2005; 280: 8929-8935Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The EPR spectral properties of the flavin semiquinone radical (FSQ) were recorded for the nNOS flavoprotein domain (20Konas D.W. Takaya N. Sharma M. Stuehr D.J. Biochemistry. 2006; 45: 12596-12609Crossref PubMed Scopus (20) Google Scholar) and for the H2B-bound, nNOS full-length protein (24Galli C. MacArthur R. AbuSoud H.M. Clark P. Stuehr D.J. Brudvig G.W. Biochemistry. 1996; 35: 2804-2810Crossref PubMed Scopus (35) Google Scholar). The FSQ radical was generated in either protein by adding a slight molar excess of NADPH to each protein in air-saturated buffer and then waiting for the flavins to air oxidize, while monitoring FSQ formation in a UV-visible spectrophotometer as a broad peak centered near 600 nm (20Konas D.W. Takaya N. Sharma M. Stuehr D.J. Biochemistry. 2006; 45: 12596-12609Crossref PubMed Scopus (20) Google Scholar). This procedure results in both proteins containing an air-stable FMN semiquinone radical (20Konas D.W. Takaya N. Sharma M. Stuehr D.J. Biochemistry. 2006; 45: 12596-12609Crossref PubMed Scopus (20) Google Scholar, 24Galli C. MacArthur R. AbuSoud H.M. Clark P. Stuehr D.J. Brudvig G.W. Biochemistry. 1996; 35: 2804-2810Crossref PubMed Scopus (35) Google Scholar). The microwave power dependence of each radical signal was analyzed using nonlinear regression in Equation 1, Log(S/P)=−b/2log(P1/2+P)+b/2log(P1/2)+log(A)(Eq. 1) where P is the power, S is the EPR intensity from double integration, P½ is the power at half-saturation, A is a scaling factor, and b is an inhomogeneity parameter. All fittings were performed using Origin Pro 7.5 (OriginLab, Northampton, MA). Determining the Concentrations of 5MeH4B and FSQ Radicals—The primary EPR spin concentration was determined using Cu(II)EDTA solutions of known concentration, and their EPR spectra recorded under conditions as described here and in earlier reports (21Wei 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, 27Hurshman A.R. Krebs C. Edmondson D.E. Huynh B.H. Marletta M.A. Biochemistry. 1999; 38: 15689-15696Crossref PubMed Scopus (212) Google Scholar). The concentration of the FSQ radical was determined based on a comparison with EPR signals from samples of the 1-electron-reduced nNOS flavoprotein domain, whose total concentration and FSQ concentration were determined by UV-visible spectroscopy using known extinction coefficients (35Konas D.W. Zhu K. Sharma M. Aulak K.S. Brudvig G.W. Stuehr D.J. J. Biol. Chem. 2004; 279: 35412-35425Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The dilution factors we used for correcting EPR signal intensities in the rapid-freeze-quench samples at each delay time were determined as done previously (21Wei 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, 25Wei C.C. Wang Z.Q. Arvai A.S. Hemann C. Hille R. Getzoff E.D. Stuehr D.J. Biochemistry. 2003; 42: 1969-1977Crossref PubMed Scopus (53) Google Scholar). Briefly, a 500 μm Cu(II)EDTA solution was rapidmixed with buffer at each delay time, subject to rapid freezing, and then the EPR signal intensities of these samples were measured. The dilution factors ranged from 2-3-fold. The EPR intensities contributed by the 5MeH4B and FSQ radical in each reaction sample were calculated using Equations 2 and 3 and assuming that (i) one heme, one 5MeH4B, one FAD, and one FMN molecule were bound in each subunit of the nNOS homodimer, and (ii) only two radical species were formed (see "Results"). I(2 milliwatts,t)=IH4B, t+IFSQ, t(Eq. 2) I(20 microwatts,t)=0.1075×IH4B, t+0.4843×IFSQ, t(Eq. 3) where I(2 milliwatts, t) and I(20 microwatts, t) were the double integraions of EPR spectra of the same sample measured at 2 milliwatts and 20 microwatts power at time t, respectively. IH4B, t and IFSQ, t are only the two variables representing the intensities of 5MeH4B and FSQ radicals at 2 milliwatts. The coefficients of 0.1075 and 0.4842 in Equation 3 were determined from power saturation measurements, indicating the fold intensity dropped from 2 milliwatts to 20 microwatts. By solving Equations 2 and 3, the EPR intensities from H4B and FSQ at time t could be determined. The 5MeH4B and FSQ radicals saturate at different microwave power: At 2 milliwatts the FSQ radical is largely saturated and no longer responsive to power, whereas the 5MeH4B radical is not saturated at 2 milliwatts. The intensities we derived from Equations 2 and 3 were then converted to the concentration of individual radicals by comparison with samples of known radical concentration prepared as described above and measured at the same power setting. For example, IH4B, t is used to determine the concentration of the 5MeH4B radical by comparison with data from Cu(II)EDTA samples of known concentrations, and IFSQ, t is used to determine the concentration of the FSQ radical by comparison of data from an authentic sample of nNOS flavoprotein FSQ radical in which its radical concentration was determined by UV-visible spectrometry. Therefore, variables in Equations 1 and 2 already embed the different power saturation behaviors of the 5MeH4B and FSQ radicals. Redox State of Bound Biopterin—The oxidation state of biopterin in CaM-bound or CaM-free nNOS samples following single catalytic turnover Arg hydroxylation reactions was determined using established methods (13Wei 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, 22Fukushima T. Nixon J.C. Anal. Biochem. 1980; 102: 176-188Crossref PubMed Scopus (636) Google Scholar) with some modifications. Approximately 70 μm nNOS protein was incubated with 0.4 mm H4B and 10 mm Arg at room temperature for 20 min and then passed through a PD-10 desalting column. Arg was then added to the protein to give a final concentration of 10 mm. A portion of the sample also received 100 μm CaM and 1 mm CaCl2, while another portion received 3 mm EDTA. The samples were made anaerobic and then reduced to ferrous by titrating with a near stoichiometric amount of dithionite solution. Approximately 0.3 ml of each anaerobic protein sample was mixed at room temperature with an equal amount of O2-saturated buffer (40 mm EPPS, pH 7.5) containing 24 μl of iodine solution (0.9% (w/v) iodine in H2O), and then was treated with standard alkaline and acidic iodine solutions used to determine concentrations of oxidized and reduced biopterin (22Fukushima T. Nixon J.C. Anal. Biochem. 1980; 102: 176-188Crossref PubMed Scopus (636) Google Scholar). After removing the precipitated protein by centrifugation, a 50-μl aliquot from each sample was then injected onto a 4.5 × 250-mm Partisil 5μ ODS-3 column (Alltech) that was equilibrated with an aqueous 5% MeOH solution. The content of H4B and its oxidation products were determined by HPLC with fluorescence detection. The percentage of H4B in the samples was calculated by subtracting the area of the peak eluting at 12 min in the aliquot that had undergone basic workup from the corresponding peak area present in the companion aliquot that had undergone acidic workup. An H4B radical forms in NOS during Arg hydroxylation to NOHA (Fig. 1, enzyme species I-III). A subsequent reduction of the H4B radical is then thought to be required (Fig. 1, enzyme species III and IV) so that NOS can oxidize the bound NOHA to NO (Fig. 1). The reversible nature of CaM binding to nNOS (16Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar, 17Abu-Soud H.M. Presta A. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10811-10816Crossref PubMed Scopus (72) Google Scholar) enabled us to create two populations of ferrous nNOS (i.e. CaM-bound and CaM-free versions of species I in Fig. 1) for use in Arg hydroxylation reactions. Because only the CaM-bound nNOS can engage in electron transfer between its flavoprotein and oxygenase domain heme (16Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar), this setup allowed us to test whether: 1) the H4B radical that forms during Arg hydroxylation is actually reduced back to H4B, 2) the nNOS flavoprotein domain provides the electron for H4B radical reduction, and 3) the electron transfer to the H4B radical requires that CaM is bound to nNOS. We utilized nNOS because its flavoprotein-to-oxygenase domain electron transfer rate (about 4 s-1 at 10 °C; Ref. 19Panda K. Adak S. Aulak K.S. Santolini J. McDonald J.F. Stuehr D.J. J. Biol. Chem. 2003; 278: 37122-37131Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) and its H4B radical formation rate during Arg hydroxylation (20 s-1 at 10 °C) are the fastest among the three NOS enzymes, while the oxidative decay of its bound H4B radical is the slowest (0.6 s-1 at 10 °C) (12Wei C.C. Wang Z.Q. Durra D. Hemann C. Hille R. Garcin E.D. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 2005; 280: 8929-8935Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 19Panda K. Adak S. Aulak K.S. Santolini J. McDonald J.F. Stuehr D.J. J. Biol. Chem. 2003; 278: 37122-37131Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). We also utilized 5MeH4B in place of H4B because it has an even faster radical formation (51 s-1) and a slower oxidative decay (0.2 s-1) in nNOS Arg hydroxylation reactions compared with H4B (12Wei C.C. Wang Z.Q. Durra D. Hemann C. Hille R. Garcin E.D. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 2005; 280: 8929-8935Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and thus provides the widest possible time window to observe reduction of the pterin radical in a NOS following Arg hydroxylation. The 5MeH4B radical also has characteristic hyperfine structure that helps to distinguish it from flavin or superoxide radical species that may also form concurrently in NOS (9Hurshman A.R. Krebs C. Edmondson D.E. Marletta M.A. Biochemistry. 2003; 42: 13287-13303Crossref PubMed Scopus (51) Google Scholar, 23Berka V. Wu G. Yeh H.C. Palmer G. Tsai A.L. J. Biol. Chem. 2004; 279: 32243-32251Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 24Galli C. MacArthur R. AbuSoud H.M. Clark P. Stuehr D.J. Brudvig G.W. Biochemistry. 1996; 35: 2804-2810Crossref PubMed Scopus (35) Google Scholar, 25Wei C.C. Wang Z.Q. Arvai A.S. Hemann C. Hille R. Getzoff E.D. Stuehr D.J. Biochemistry. 2003; 42: 1969-1977Crossref PubMed Scopus (53) Google Scholar). In general, the reactions were initiated at 10 °C by rapidly mixing an O2 saturated buffer (∼1.7 mm O2) with an anaerobic solution of NADPH pre-reduced CaM-bound or CaM-free ferrous nNOS (∼0.12 mm) that contained saturating concentrations of Arg and 5MeH4B. Following mixing, the reaction samples were aged for different times within the instrument and then underwent rapid freezing. The frozen reaction samples then had their free radical spectra and content analyzed by EPR. This procedure allowed us to study the sequential reaction steps I through IV (Fig. 1) during the Arg hydroxylation reaction catalyzed by nNOS. Under our reaction conditions, O2 binding to the ferrous heme is rapid compared with subsequent H4B radical formation and Arg hydroxylation, as indicated by the rates measured for these steps under near-identical reaction conditions (Fig. 1 and Refs. 12Wei C.C. Wang Z.Q. Durra D. Hemann C. Hille R. Garcin E.D. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 2005; 280: 8929-8935Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 21Wei 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, 25Wei C.C. Wang Z.Q. Arvai A.S. Hemann C. Hille R. Getzoff E.D. Stuehr D.J. Biochemistry. 2003; 42: 1969-1977Crossref PubMed Scopus (53) Google Scholar). In our reactions we expect that EPR signals from the bound 5MeH4B radical and from the FSQ radical will be present simultaneously during the course of the Arg hydroxylation reaction. Fig. 2 shows that this was the case. The figure contains representative EPR traces of reaction samples that were aged for the indicated times during an Arg reaction catalyzed by either CaM-free (panel A) or CaM-bound (panel B) nNOS. Standard EPR spectra of the 5MeH4B radical and the nNOS FSQ radical are included in Fig. 2 for comparison. At 80 and 125 ms, the EPR traces have peak-to-trough values that range from 32 to 39 G. Because this value is close to the peak-to-trough value for authentic 5MeH4B radical (40 G) and the traces otherwise display characteristic spectral features and hyperfine structure of the 5MeH4B radical (9Hurshman A.R. Krebs C. Edmondson D.E. Marletta M.A. Biochemistry. 2003; 42: 13287-13303Crossref PubMed Scopus (51) Google Scholar, 25Wei C.C. Wang Z.Q. Arvai A.S. Hemann C. Hille R. Getzoff E.D. Stuehr D.J. Biochemistry. 2003; 42: 1969-1977Crossref PubMed Scopus (53) Google Scholar), it appears to be the dominant radical present in the enzyme reaction samples at these two early time points. For the 1- and 2-s reaction samples, however, the peak-to-trough width declines to approach the value of the nNOS FSQ radical (20 G), and the spectral traces lose characteristic features that indicate a disappearance of the 5MeH4B radical, particularly in the reaction catalyzed by the CaM-bound nNOS (panel B). The radical signals in these traces indicate the FSQ radical appears to become predominant in both reactions by 2 s (24Galli C. MacArthur R. AbuSoud H.M. Clark P. Stuehr D.J. Brudvig G.W. Biochemistry. 1996; 35: 2804-2810Crossref PubMed Scopus (35) Google Scholar, 26Perry J.M. Moon N. Zhao Y. Dunham W.R. Marletta M.A. Chem. Biol. 1998; 5: 355-364Abstract Full Text PDF PubMed Scopus (19) Google Scholar). Thus, a qualitative analysis of the traces suggest that the 5MeH4B radical builds up and then begins to disappear within the 0 to 2 s reaction timeframe, concurrent with a more gradual and sustained b
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