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• W1973695591 abstract "Characterization of the redox properties of endothelial nitric-oxide synthase (eNOS) is fundamental to understanding the complicated reaction mechanism of this important enzyme participating in cardiovascular function. Yeast overexpression of both the oxygenase and reductase domains of human eNOS,i.e. eNOSox and eNOSred, has been established to accomplish this goal. UV-visible and electron paramagnetic resonance (EPR) spectral characterization for the resting eNOSox and its complexes with various ligands indicated a standard NOS heme structure as a thiolate hemeprotein. Two low spin imidazole heme complexes but not the isolated eNOSox were resolved by EPR indicating slight difference in heme geometry of the dimeric eNOSox domain. Stoichiometric titration of eNOSox demonstrated that the heme has a capacity for a reducing equivalent of 1–1.5. Additional 1.5–2.5 reducing equivalents were consumed before heme reduction occurred indicating the presence of other unknown high potential redox centers. There is no indication for additional metal centers that could explain this extra electron capacity of eNOSox. Ferrous eNOSox, in the presence of l-arginine, is fully functional in forming the tetrahydrobiopterin radical upon mixing with oxygen as demonstrated by rapid-freeze EPR measurements. Calmodulin binds eNOSred at 1:1 stoichiometry and high affinity. Stoichiometric titration and computer simulation enabled the determination for three redox potential separations between the four half-reactions of FMN and FAD. The extinction coefficient could also be resolved for each flavin for its semiquinone, oxidized, and reduced forms at multiple wavelengths. This first redox characterization on both eNOS domains by stoichiometric titration and the generation of a high quality EPR spectrum for the BH4 radical intermediate illustrated the usefulness of these tools in future detailed investigations into the reaction mechanism of eNOS. Characterization of the redox properties of endothelial nitric-oxide synthase (eNOS) is fundamental to understanding the complicated reaction mechanism of this important enzyme participating in cardiovascular function. Yeast overexpression of both the oxygenase and reductase domains of human eNOS,i.e. eNOSox and eNOSred, has been established to accomplish this goal. UV-visible and electron paramagnetic resonance (EPR) spectral characterization for the resting eNOSox and its complexes with various ligands indicated a standard NOS heme structure as a thiolate hemeprotein. Two low spin imidazole heme complexes but not the isolated eNOSox were resolved by EPR indicating slight difference in heme geometry of the dimeric eNOSox domain. Stoichiometric titration of eNOSox demonstrated that the heme has a capacity for a reducing equivalent of 1–1.5. Additional 1.5–2.5 reducing equivalents were consumed before heme reduction occurred indicating the presence of other unknown high potential redox centers. There is no indication for additional metal centers that could explain this extra electron capacity of eNOSox. Ferrous eNOSox, in the presence of l-arginine, is fully functional in forming the tetrahydrobiopterin radical upon mixing with oxygen as demonstrated by rapid-freeze EPR measurements. Calmodulin binds eNOSred at 1:1 stoichiometry and high affinity. Stoichiometric titration and computer simulation enabled the determination for three redox potential separations between the four half-reactions of FMN and FAD. The extinction coefficient could also be resolved for each flavin for its semiquinone, oxidized, and reduced forms at multiple wavelengths. This first redox characterization on both eNOS domains by stoichiometric titration and the generation of a high quality EPR spectrum for the BH4 radical intermediate illustrated the usefulness of these tools in future detailed investigations into the reaction mechanism of eNOS. Nitric-oxide synthase (NOS) 1The abbreviations used are: NOS, nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; eNOSox, eNOS oxygenase domain; eNOSred, eNOS reductase domain; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; BH2, dihydrobiopterin; BH4, (6R)-5,6,7,8-tetrahydro-l-biopterin; BCA, bicinchoninic acid; EPR, electron paramagnetic resonance; ICP-MS, inductively coupled plasma emission mass spectrometry; CPR, cytochrome P450 reductase; CaM, calmodulin; DCPIP, 2,6-dichlorophenol indophenol is an uncommon self-sufficient P450-like enzyme catalyzing nitric oxide (NO) biosynthesis from l-arginine (1Roman L.J. Martásek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1189Google Scholar, 2Raman C.S. Martásek P. Masters B.S.S. Kadish K.M. Smith K.M. Guilard R. The Porphyrin Handbook. Academic Press, New York2000: 293-327Google Scholar, 3Feldman P.L. Griffith O.W. Stuehr D.J. Chem. Eng. News. 1993; 71: 26-38Google Scholar, 4Alderton W.K. Cooper C.E. Knowles R.G. Biochem. J. 2000; 357: 593-615Google Scholar). There are three mammalian NOS isozymes: the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) require calmodulin for enzyme activity, whereas the inducible NOS (iNOS) contains tightly bound calmodulin (1Roman L.J. Martásek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1189Google Scholar, 2Raman C.S. Martásek P. Masters B.S.S. Kadish K.M. Smith K.M. Guilard R. The Porphyrin Handbook. Academic Press, New York2000: 293-327Google Scholar, 3Feldman P.L. Griffith O.W. Stuehr D.J. Chem. Eng. News. 1993; 71: 26-38Google Scholar, 4Alderton W.K. Cooper C.E. Knowles R.G. Biochem. J. 2000; 357: 593-615Google Scholar). All three isozymes have a common bi-domain structure with the reductase domain containing FAD, FMN, and NADPH binding sites, and the oxygenase domain harboring the heme center and binding sites forl-arginine and tetrahydrobiopterin (BH4) (1Roman L.J. Martásek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1189Google Scholar, 2Raman C.S. Martásek P. Masters B.S.S. Kadish K.M. Smith K.M. Guilard R. The Porphyrin Handbook. Academic Press, New York2000: 293-327Google Scholar, 3Feldman P.L. Griffith O.W. Stuehr D.J. Chem. Eng. News. 1993; 71: 26-38Google Scholar, 4Alderton W.K. Cooper C.E. Knowles R.G. Biochem. J. 2000; 357: 593-615Google Scholar). The main function of the reductase domain is to provide reducing equivalents to the heme center in the oxygenase domain where the key chemistry of l-arginine conversion occurs. Three substrates and four products are involved in NOS catalysis. The overall reaction is a complicated five-electron oxidation of the key guanidine nitrogen plus three additional electrons from NADPH to reduce two molecules of oxygen to water and form the l-citrulline and nitric oxide. Several x-ray crystallographic structures for the iNOS and eNOS oxygenase domains have been reported (5Raman C.S. Li H. Martásek P. Král V. Masters B.S.S. Poulos T.L. Cell. 1998; 95: 1-20Google Scholar, 6Fischmann T.O. Hruza A. Xiao D.N. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Reichert P. Lundell D.J. Narula S.K. Weber P.C. Nat. Struct. Biol. 1999; 6: 233-242Google Scholar, 7Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Google Scholar). The x-ray crystallographic data at 1.9-Å resolution of the C-terminal FAD-NADPH binding domain of the nNOS reductase domain was also published recently (8Zhang J. Martásek P. Paschke R. Shea T. Masters B.S.S. Kim J.J. J. Biol. Chem. 2001; 276: 37506-37513Google Scholar). These data reveal a three-domain modular design. The FAD and NADPH binding subdomains are superimposable on those of cytochrome P450 reductase (CPR) with a root mean square deviation of 1.3 Å, whereas the more flexible FMN-connecting domain shows a 3.9-Å root mean square deviation to the α-chain of CPR. The fourth domain that binds FMN is lost during crystallization, but the structure is projected to be similar to that of CPR. These crystallographic data give firm support for a modular design of NOS and thus provide a basis to prepare subdomains for structure/function and reaction mechanism studies. Investigation into individual breakdown modules could simplify the data interpretation for each redox center and should be a useful approach in elucidation of the complicated reaction mechanism for NOS. Overexpression systems for the individual oxygenase and reductase domain of NOS have been developed in bacterial and baculovirus systems, including our own group (9Hurshman A.R. Marletta M.A. Biochemistry. 2002; 41: 3439-3456Google Scholar, 10Perry J.M. Moon N. Zhao Y. Dunham W.R. Marletta M.A. Chem. Biol. 1998; 5: 355-364Google Scholar, 11McMillan K. Masters B.S. Biochemistry. 1995; 34: 3686-3693Google Scholar, 12Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Google Scholar, 13Ghosh D.K. Stuehr D.J. Biochemistry. 1995; 34: 801-807Google Scholar, 14Gachhui R. Presta A. Bentley D.F. Abu-Soud H.M. McArthur R. Brudvig G. Ghosh D.K. Stuehr D.J. J. Biol. Chem. 1996; 271: 20594-20602Google Scholar, 15Bec N. Gorren A.C.F. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Google Scholar, 16Mayer B. Klatt P. Harteneck C. List B.M. Werner E.R. Schmidt K. Methods Neurosci. 1996; 31: 130-139Google Scholar, 17Chen P.-F. Tsai A.-L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Google Scholar). Only a few are related to eNOS (6Fischmann T.O. Hruza A. Xiao D.N. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Reichert P. Lundell D.J. Narula S.K. Weber P.C. Nat. Struct. Biol. 1999; 6: 233-242Google Scholar, 17Chen P.-F. Tsai A.-L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Google Scholar). Large amounts of eNOSox were usually obtained by trypsinolysis from intact bovine eNOS (13Ghosh D.K. Stuehr D.J. Biochemistry. 1995; 34: 801-807Google Scholar, 18Martásek P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Google Scholar). Although the baculovirus system is useful (17Chen P.-F. Tsai A.-L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Google Scholar), it is both time-consuming and costly. The bacterial expression system (18Martásek P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Google Scholar), although fast, has unpredictable sudden debilitating mutations in the expression construct and, in our hands, has resisted being scaled up to more than a few liters of culture for unknown reasons. Yeast expression could be an alternative vehicle to generate large amounts of active mammalian enzymes (19Romanos M.A. Scorer C.A. Clare J.J. Yeast. 1992; 8: 423-488Google Scholar). Yeast has been shown to be effective in overexpressing eNOS and the reductase domain of nNOS (14Gachhui R. Presta A. Bentley D.F. Abu-Soud H.M. McArthur R. Brudvig G. Ghosh D.K. Stuehr D.J. J. Biol. Chem. 1996; 271: 20594-20602Google Scholar, 20Leber A. Hemmens B. Klosch B. Goessler W. Raber G. Mayer B. Schmidt K. J. Biol. Chem. 1999; 274: 37658-37664Google Scholar). Here, we report the overexpression in yeast the oxygenase, eNOSox, and reductase, eNOSred, domains of human eNOS and the characterization for their oxidation-reduction activities. Both domains show behaviors very similar to the domains present in the whole eNOS and should be useful tools for future biophysical and mechanistic investigations. BH4 was obtained from Schircks Laboratories (Jona, Switzerland). Plasmids containing human eNOS cDNA in pGEM3Z and human eNOS polyclonal antibodies were kindly provided by Dr. Pei-Feng Chen in our division (21Chen P.-F. Tsai A.-L. Wu K.K. Biochem. Biophys. Res. Commun. 1995; 215: 1119-1129Google Scholar). PCR kits (Expand High Fidelity PCR system) were the product of Roche Molecular Biochemicals. Restriction enzyme PmeI was purchased from New England BioLabs, and the other restriction enzymes were from Invitrogen. All reagents and devices for DNA extraction and isolation were products of Qiagen. An Easyselect Pichia Expression kit, containing the expression vector pPICZB, Pichia strain GS115, and Escherichia coil strain TOP10F′, was purchased from Invitrogen and used for the expression of both eNOS domains. Reagents for electrophoresis and Western blotting were from Bio-Rad. The remaining chemicals were from Sigma. PCR was used to amplify the cDNA product. Human eNOS cDNA in pGEM3Z was used as template, and DNA fragments encoding oxygenase (amino acids 1–491) and reductase domain (amino acids 482–1204) were amplified with specific primers. For oxygenase domain, the forward primer was 5′-CGGAATTCAACATGCATCACCATCACCATCACGGCAACTTGAAGAGCGTG-3′ (translation start codon is underlined), and the backward primer was 5′-GCTCTAGATCAGGTGATGCCGGTGCCCTTGGC-3′ (translation stop codon is underlined). For reductase domain, the forward primer was 5′-CGGAATTCAACATGCATCACCATCACCATCACGGGAGTGCCGCCAAGGGC-3′, and the backward primer was 5′-GCTCTAGATCAGGGGCTGTTGGTGTCTGAGCC-3′. In both forward primers, the EcoRI site and His6 tag were added, and in each backward primer an XbaI site was added. The correct sequences of the PCR products were confirmed by primer extension sequencing. Both PCR products were double-digested with EcoRI and XbaI and subcloned separately into the corresponding sites of an alcohol oxidase promoter-driven expression vector pPICZB to obtain a 1.5-kb insert of eNOSox and a 2.1-kb insert of eNOSred. The constructs were linearized with PmeI, transformed into yeastPichia pastoris GS115, and selected by growing them on the YPDS/Zeocin plates containing 1% yeast extract, 2% peptone, 2% dextrose, 1 m sorbitol, and 100 μg/ml Zeocin. The colony that grew fastest was inoculated into 25 ml of buffered minimal glycerol medium (100 mm potassium phosphate, pH 6.0, 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base with ammonium sulfate without amino acids, 4 × 10−5% biotin and 1% glycerol) and cultured at 30 °C overnight. This culture was then transferred to a 250-ml buffered minimal glycerol medium and grown at 30 °C overnight to A 600 = 7–10. Cells were harvested and resuspended in 250 ml of buffered minimal methanol medium (100 mm potassium phosphate, pH 6.0, 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base with ammonium sulfate without amino acids, 4 × 10−5% biotin, and 0.5% methanol) and cultured for 72 h at 30 °C to induce protein expression. Yeast cells were harvested and washed with buffer 1 (50 mm Tris-HCl, pH 8.0) with protease inhibitors (1 μm leupeptin, 1 μm antipain, 1 μm pepstatin A, and 1 mmphenylmethylsulfonyl fluoride) and resuspended in an equal volume of buffer 1 with protease inhibitors. An equal volume of glass beads (425–600 μm) was added to the suspension. Cells were broken by 10 cycles of 30-s vortexing and brief chilling on ice. Cell debris and glass beads were removed by centrifugation at 3,400 rpm. The supernatant, obtained after another centrifugation at 12,000 rpm in a microcentrifuge, was applied to a 2-ml nickel-nitrilotriacetic acid-agarose column. The column was first washed with a 50-bed volume of buffer 1 plus protease inhibitors, then washed with a ∼30-bed volume of buffer 1 plus 0.3 m NaCl and 1 mml-histidine, then by a ∼20-bed volume buffer 1 plus 0.1 m NaCl and 5 mml-histidine. Finally, buffer 1 plus 40 mml-histidine was used to elute bound oxygenase domain and 100 mml-histidine for the reductase domain. The eluate was concentrated by Centriprep-50 then applied to a 10-DG column (Bio-Rad) and eluted with 50 mm HEPES, pH 7.4, containing 0.1 m NaCl and 10% glycerol, to remove histidine. The content of BH4, FAD, and FMN of purified eNOS domains was measured as described previously (17Chen P.-F. Tsai A.-L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Google Scholar, 21Chen P.-F. Tsai A.-L. Wu K.K. Biochem. Biophys. Res. Commun. 1995; 215: 1119-1129Google Scholar) and quantified from a standard curve of authentic BH4, FAD, or FMN, respectively. Biopterin determination was done on eNOSox with or without reconstitution with exogenous BH4. BH4reconstitution was done similarly to procedures that were published previously (9Hurshman A.R. Marletta M.A. Biochemistry. 2002; 41: 3439-3456Google Scholar, 22Rusche K.M. Marletta M.A. J. Biol. Chem. 2001; 276: 421-427Google Scholar) under anaerobic condition. The excess amount of BH4 was removed by gel filtration, and the amount of bound BH4 was determined using our HPLC quantitation similar to the published procedure using authentic BH4 to build a standard curve (23Lunte C.E. Kissinger P.T. Anal. Biochem. 1983; 129: 377-386Google Scholar). Heme content was determined by the formation of pyridine hemochromogen as previously described (24Berka V. Palmer G. Chen P.-F. Tsai A.-L. Biochemistry. 1998; 37: 6136-6144Google Scholar). The total heme content was determined from difference spectrum of bis-pyridine heme (reduced minus oxidized) using Δε556–538 nm = 24 mm−1cm−1. Surface-exposed thiol groups were determined by chemical modification using 4,4′-dithiopyridine to form a 4-thiopyridone chromophore with major absorbance at 343 nm. The 4,4′-dithiopyridine itself has almost no absorption at that wavelength (25Grassetti D.R. Murray Jr., J.F. J. Chromatogr. 1996; 41: 121-123Google Scholar). Cytochrome creductase activity was measured as the absorbance increase at 550 nm using Δε = 21 mm−1 cm−1as described previously (17Chen P.-F. Tsai A.-L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Google Scholar). Ferricyanide or 2,6-dichlorophenol indolphenol oxidation assay was carried out using Δε = 1 mm−1 cm−1 at 400 nm and Δε = 21 mm−1 cm−1 at 600 nm, respectively (14Gachhui R. Presta A. Bentley D.F. Abu-Soud H.M. McArthur R. Brudvig G. Ghosh D.K. Stuehr D.J. J. Biol. Chem. 1996; 271: 20594-20602Google Scholar). This activity measurement essentially followed previous published procedure for iNOSox (26Hurshman A.R. Krebs C. Edmondson D.E. Huynh B.H. Marletta M.A. Biochemistry. 1999; 38: 15689-15696Google Scholar, 27Wei C.C. Wang Z.Q. Wang Q. Meade A.L. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2001; 276: 315-319Google Scholar). High concentration of BH4-reconstituted eNOSox was reduced anaerobically in a tonometer by dithionite titration in the presence of 1 mm l-arginine. The ferrous eNOSox was then reacted with oxygenated buffer using a rapid-freeze/EPR technique as we previously published (28Tsai A.-L. Wu G. Palmer G. Bambai B. Koehn J.A. Marshall P.J. Kulmacz R.J. J. Biol. Chem. 1999; 274: 21695-21700Google Scholar, 34Tsai A.-L. Berka V. Kulmacz R.J. Wu G. Palmer G. Anal. Biochem. 1998; 264: 165-171Google Scholar). The rapid-freeze apparatus, System 1000 (Update Instrument, Madison, WI), was placed inside an anaerobic chamber (Coy Laboratory). The oxygen level was lower than 5 ppm during the whole experiment procedure and monitored by an oxygen/hydrogen analyzer (Model 10, Coy Laboratory). One or two push programs were used to obtain samples freeze-trapped at different reaction times. UV-visible spectra were measured on an HP8453 diode array spectrophotometer with a 1-nm spectral bandwidth. EPR results were recorded at liquid helium or liquid nitrogen temperature on a Bruker EMX EPR spectrometer. For liquid helium system, a GFS600 transfer line and an ITC503 temperature controller were used to maintain the temperature. An Oxford ESR900 cryostat was used to accommodate the sample. For liquid nitrogen transfer, a silver-coated double-jacketed glass transfer line and a BVT3000 temperature controller were used. Data analysis was conducted using WinEPR, and spectral simulations were done using SimFonia programs provided by Bruker. Flavin fluorescence was measured using an SLM SPF-500C spectrofluorometer using the ratio mode. About 2 μmeNOSred in a 1-cm quartz cuvette was excited at 450 nm (5-nm spectral bandwidth), and the emission spectrum between 450 and 650 nm (7.5-nm spectral bandwidth) was collected at 24 °C. The redox capacities of eNOSred and eNOSox were determined by anaerobic stoichiometric titration using sodium dithionite. Stock solution of sodium dithionite was freshly prepared by dissolving powdered reagent in 50 mm, pH 8.2 pyrophosphate buffer pre-saturated with pure nitrogen gas. The concentration of sodium dithionite was standardized by titration against a fixed amount of lumiflavin-3-acetic acid (ε444 = 1.08 × 104m−1cm−1) anaerobically before and after individual real sample titration (29Foust G.P. Burleigh Jr., B.D. Mayhew S.G. Williams D.H. Massey V. Anal. Biochem. 1969; 27: 530-535Google Scholar). The average concentration was used to calculate the number of reducing equivalents consumed in the titrations. Each protein sample was placed in an anaerobic titrator and made anaerobic by 5 cycles of evacuation (30 s) and argon replacement (5 min). Standardized dithionite solution contained in a gas-tight syringe engaged to the side arm of the titrator was quantitatively delivered and mixed with the protein sample under argon atmosphere. The electronic spectrum was recorded on an HP8452 diode array spectrophotometer to confirm that the system was equilibrated after each addition of dithionite reflected by a static absorbance. The protein content was determined by BCA method (30Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Google Scholar). SDS-PAGE was performed on 10% Ready-Gels in a Bio-Rad mini-gel apparatus. Gel filtration chromatography was performed on a Sephacryl 200 HR column (1.5 × 50 cm). A kit for molecular weight 12,000–200,000 (product code: MW-GF-200) was used as the gel filtration marker. The SCoP program (Simulation Resources Inc., Redlands, CA) was used for simulating the data obtained from stoichiometric titration, mainly the eNOSred similar to the method used by Iyanagi et al. (31Iyanagi T. Makino N. Mason H.S. Biochemistry. 1974; 13: 1701-1710Google Scholar). The absorbance changes at different monitoring wavelengths during titration were simulated against accumulated reducing equivalents added, Aλ=ɛ1×F1+ɛ2×F1H+ɛ3×F1H2+ɛ4×F2+ɛ5×F2H+ɛ6×F2H2(Eq. 1) where A λ is the observed absorbance at wavelength λ, and ε1 through ε6 are the extinction coefficients for each flavin redox species (F1, F1H, and F1H2 represent fully oxidized, semiquinone, and fully reduced forms of the first flavin, respectively, and F2, F2H, and F2H2 are the equivalents for the second flavin). The concentrations of each flavin intermediate during a stoichiometric titration are expressed as follows, F1=[(x1×x2)/(1+x2+x1+x2)]×f1(Eq. 2) F1H=[x2/(1+x2+x1×x2)]×f1(Eq. 3) F1H2=[1/(1+x2+x1×x2)]×f1(Eq. 4) F2=[(x3×x4)/(1+x4+x3×x4)]×f2(Eq. 5) F2H=[x4/(1+x4+x3×x4)]×f2(Eq. 6) F2H2=[1/(1+x4+x3×x4)]×f2(Eq. 7) where f1 and f2 are the total amounts of each flavin and, x1=exp{[F/(R×T)]×(Eh–E1)}(Eq. 8) x2=exp{[F/(R×T)]×(Eh–E2)}(Eq. 9) x3=exp{[F/(R×T)]×(Eh–E3)}(Eq. 10) x4=exp{[F/(R×T)]×(Eh–E4)}(Eq. 11) where E1 through E4 are midpoint potentials of the four half-reactions of the two flavins in eNOSred, Eh is any measured redox potential value, F is the Faraday constant (96,485 Coulomb mol−1), R is the gas constant (8.314 J K−1mol−1), and T is temperature (298 K). The total reducing equivalents were simply expressed as, Equivalents=2×[(F1H+2×F1H2+F2H+2×F2H2)/ft](Eq. 12) Where f t is the total flavin,i.e. the sum of f1 and f2. Simulation was generated by sweeping the Eh values in any desired potential range and seeking optimal values forE1–E4 to achieve the best fit to the observed data. It is not possible to achieve a set of absolute midpoint potentials, but the relative midpoint potential values can be converged. In other words, once one of the E1–E4 values is fixed, the other three can be located by simulation. Absorbance extinction coefficients for the fully oxidized and fully reduced flavins are readily available, and those for the flavin semiquinone can be properly estimated from the spectrum at the stage of one- and three-electron-reduced states. We also let these two coefficients be variable in a narrow range and optimized their values via simulation. The yeast expression vector containing the AOX promoter is promising in overexpressing both domains of eNOS in P. pastoris. By introducing a His6 tag in the N terminus of both domains, purification of the target protein can be conveniently done by nickel-nitrilotriacetic acid-agarose column chromatography. The average yields of the purified oxygenase domain and the reductase domain are ∼8 and ∼22 mg/liter, respectively. Both purified eNOSoxand eNOSred resulted in a single band on SDS-PAGE with apparent molecular masses of 54 and 82 kDa, respectively (Fig.1 A) and exhibited immunoreactivity with polyclonal antibodies against eNOS (Fig.1 B). The heme content determined by pyridine hemochromogen assay was 0.93 ± 0.04 (n = 7), almost stoichiometric to the protein subunit (Table I). Replenishing hemin or δ-aminolevulinic acid to the cell medium during yeast growth did not further increase the heme content in purified eNOSox. The purified oxygenase domain also contained endogenous biopterin at a stoichiometry lower than 0.3/monomer. Because our sample buffers did not include dithiothreitol, most of these biopterin molecules were present as dihydrobiopterin, BH2, as analyzed by our HPLC method (data not shown). The functional form of biopterin, BH4, can be reconstituted back to the purified eNOSox according to the anaerobic procedure similar to that described by Rusche and Marletta (22Rusche K.M. Marletta M.A. J. Biol. Chem. 2001; 276: 421-427Google Scholar). The reconstituted eNOSox has biopterin content as high as 0.72 per monomer (Table I) and is present as the fully reduced form, BH4, as analyzed by our HPLC method (data not shown).Table IStoichiometry of the cofactors of purified eNOS subdomains (n = 7)HemeFADFMNBH40.93 ± 0.04aThis study, for eNOSox.1.0 ± 0.33bThis study, for eNOSred.1.14 ± 0.21bThis study, for eNOSred.0.72 ± 0.12aThis study, for eNOSox.0.85 ± 0.09 (A 398)cData from Ref. 20, determined for whole eNOS.0.56 ± 0.060.79 ± 0.08NDdND, not determined.0.71 ± 0.02 (HPLC)a This study, for eNOSox.b This study, for eNOSred.c Data from Ref. 20Leber A. Hemmens B. Klosch B. Goessler W. Raber G. Mayer B. Schmidt K. J. Biol. Chem. 1999; 274: 37658-37664Google Scholar, determined for whole eNOS.d ND, not determined. Open table in a new tab The content of both FAD and FMN in purified eNOSred is essentially stoichiometric based on our HPLC determination against authentic FAD and FMN standards (Table I). The ratios of FAD and FMN to that of the eNOSred monomer are 1.0 and 1.14, respectively, thus further reconstitution of flavins is unnecessary. UV-visible spectral analyses from 250 to 700 nm of purified eNOSox showed a Soret peak at 400–404 nm, 81 mm−1cm−1, a broad α/β band at 518 nm, 15.4 mm−1cm−1, and a charge-transfer band at 645 nm, 5.8 mm−1cm−1. Treatment withl-arginine shifted the Soret peak to 396 nm with comparable amplitude, 82 mm−1cm−1, and only slight changes in the visible region (Fig.2). When eNOSox was reduced by dithionite, the Soret band is red-shifted to 413 nm with a sizable decrease in intensity, 66.7 mm−1cm−1. The α/β band also shifted to 552 nm, 13.0 mm−1cm−1, and the charge-transfer band at about 650 nm was abolished as the lower lying three metal d-orbitals were completely filled. Further addition of CO resulted in the hallmark 444-nm Soret band for P450 hemeproteins with an extinction of 91.3 mm−1cm−1 with the features found at the visible region very similar to that of ferrous eNOSox. These spectral behaviors are very similar to our bacterial-expressed eNOSox and other NOS oxygenase domains (9Hurshman A.R. Marletta M.A. Biochemistry. 2002; 41: 3439-3456Google Scholar, 13Ghosh D.K. Stuehr D.J. Biochemistry. 1995; 34: 801-807Google Scholar, 17Chen P.-F. Tsai A.-L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Google Scholar). The ratio of 280 nm to the Soret peak to either the resting or l-arginine-treated eNOSox was ∼1.5, which is an index of the purity of the hemeprotein and is a reliable number compared with other NOS preparations (9Hurshman A.R. Marletta M.A. Biochemistry. 2002; 41: 3439-3456Google Scholar, 13Ghosh D.K. Stuehr D.J. Biochemistry. 1995; 34: 801-807Google Scholar,17Chen P.-F. Tsai A.-L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Google Scholar). Liquid helium temperature EPR of the resting eNOSox showed a mixture of high spin and low spin heme structures (Fig.3, spectrum A). The rhombic high spin heme has g values of 7.53, 4.23, and 1.83 (theg min was only observable at somewhat lower temperature, ∼ 4 K), and the low spin heme show conspicuous rhombicg values at 2.43, 2.28, and 1.90. Both sets of parameters are typical for NOS and other P450 type hemeproteins containing a cysteine thiolate proximal heme ligand (32Stuehr D.J. Ikeda-Saito M. J. Biol. Chem. 1992; 267: 20547-20550Google Scholar, 33Tsai A.-L. Berka V. Chen P.-F. Palmer G. J. Biol. Chem. 1996; 271: 32563-32571Google Scholar). Addition of excess amounts of l-arginine essentially wiped out the low spin heme signals and substantially increased the high spin heme signals (Fig. 3, spectrum B). The g values of the high spin heme shifted to 7.56, 4.17, and 1.82, corresponding to a small rhombicity shift from 20.6 to 21.1%. On the other hand, imidazole converted eNOSox to fully low spin heme complex (Fig. 3,spectrum C). There are two well-resolved rhombic low spin heme complexes with g values of 2.71/2.29/1.75 and 2.60/2.29/1.81. Resolution in the EPR spectrum of the two imidazole low spin heme was even better than that found for whole eNOS (33Tsai A.-L. Berka V. Chen P.-F. Palmer G. J. Biol. Chem. 1996; 271: 32563-32571Google Scholar). To determine the redox capacity of the purified eNOSox, a stoichiometric" @default.
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• W1973695591 title "Redox Properties of Human Endothelial Nitric-oxide Synthase Oxygenase and Reductase Domains Purified from Yeast Expression System" @default.
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