Redox Properties of Human Endothelial Nitric-oxide Synthase Oxygenase and Reductase Domains Purified from Yeast Expression System*

Mei DuDagger, Hui-Chun YehDagger, Vladimir BerkaDagger, Lee-Ho Wang, and Ah-lim Tsai§

From the Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas 77030

Received for publication, September 19, 2002, and in revised form, December 3, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nitric-oxide synthase (NOS)1 is an uncommon self-sufficient P450-like enzyme catalyzing nitric oxide (NO) biosynthesis from L-arginine (1-4). 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 (1-4). 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 for L-arginine and tetrahydrobiopterin (BH4) (1-4). 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 (5-7). 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 (8). 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 alpha -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 (9-17). Only a few are related to eNOS (6, 17). Large amounts of eNOSox were usually obtained by trypsinolysis from intact bovine eNOS (13, 18). Although the baculovirus system is useful (17), it is both time-consuming and costly. The bacterial expression system (18), 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 (19). Yeast has been shown to be effective in overexpressing eNOS and the reductase domain of nNOS (14, 20). 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 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 (21). 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.

Expression of Human eNOS Domains-- 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 yeast Pichia 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 A600 = 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.

Protein Purification-- 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 mM phenylmethylsulfonyl 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 mM L-histidine, then by a ~20-bed volume buffer 1 plus 0.1 M NaCl and 5 mM L-histidine. Finally, buffer 1 plus 40 mM L-histidine was used to elute bound oxygenase domain and 100 mM L-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.

Biopterin and Flavin Determination-- The content of BH4, FAD, and FMN of purified eNOS domains was measured as described previously (17, 21) 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. BH4 reconstitution was done similarly to procedures that were published previously (9, 22) 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 (23).

Pyridine Hemochromogen Assay-- Heme content was determined by the formation of pyridine hemochromogen as previously described (24). The total heme content was determined from difference spectrum of bis-pyridine heme (reduced minus oxidized) using Delta epsilon 556-538 nm = 24 mM-1 cm-1.

Quantification of Thiol Functional Groups-- 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 (25).

eNOSred Activity Assay-- Cytochrome c reductase activity was measured as the absorbance increase at 550 nm using Delta epsilon  = 21 mM-1 cm-1 as described previously (17). Ferricyanide or 2,6-dichlorophenol indolphenol oxidation assay was carried out using Delta epsilon  = 1 mM-1 cm-1 at 400 nm and Delta epsilon  = 21 mM-1 cm-1 at 600 nm, respectively (14).

eNOSox Activity in Generating Biopterin Radical-- This activity measurement essentially followed previous published procedure for iNOSox (26, 27). 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 (28, 34). 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.

Spectrometry-- 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 µM eNOSred 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.

Stoichiometric Titration-- 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 (epsilon 444 = 1.08 × 104 M-1cm-1) anaerobically before and after individual real sample titration (29). 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.

Miscellaneous Methods-- The protein content was determined by BCA method (30). 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.

Computer Modeling-- 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. (31). The absorbance changes at different monitoring wavelengths during titration were simulated against accumulated reducing equivalents added,
A<SUB>&lgr;</SUB><UP>=ϵ1 × F1+ϵ2 × F1H+ϵ3 × F1H<SUB>2</SUB>+ϵ4 × F2+ϵ5 × F2H+ϵ6 × F2H<SUB>2</SUB></UP> (Eq. 1)
where Alambda is the observed absorbance at wavelength lambda , and epsilon 1 through epsilon 6 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,
<UP>F</UP>1=[(x<UP>1 × </UP>x<UP>2</UP>)<UP>/</UP>(<UP>1+</UP>x<UP>2</UP>+x<UP>1</UP>+x<UP>2</UP>)]<UP> × f1</UP> (Eq. 2)

<UP>F1H</UP>=[x<UP>2/</UP>(<UP>1+</UP>x<UP>2+</UP>x<UP>1</UP>×x<UP>2</UP>)]<UP> × f1</UP> (Eq. 3)

<UP>F1H</UP><SUB>2</SUB>=[1/(1+x<UP>2+</UP>x<UP>1</UP>×x<UP>2</UP>)]<UP> × f1</UP> (Eq. 4)

<UP>F2</UP>=[(x<UP>3 × </UP>x<UP>4</UP>)<UP>/</UP>(<UP>1+</UP>x<UP>4+</UP>x<UP>3 × </UP>x<UP>4</UP>)]<UP> × f2</UP> (Eq. 5)

<UP>F2H</UP>=[x<UP>4/</UP>(<UP>1+</UP>x<UP>4+</UP>x<UP>3 × </UP>x<UP>4</UP>)]<UP> × f2</UP> (Eq. 6)

<UP>F2H</UP><SUB>2</SUB>=[1/(1+x<UP>4+</UP>x<UP>3 × </UP>x<UP>4</UP>)]<UP> × f2</UP> (Eq. 7)
where f1 and f2 are the total amounts of each flavin and,
x<UP>1=exp</UP>{[F/(R×T)]×(Eh–E1)} (Eq. 8)

x<UP>2=exp</UP>{[F/(R×T)]×(Eh–E2)} (Eq. 9)

x<UP>3=exp</UP>{[F/(R×T)]×(Eh–E3)} (Eq. 10)

x<UP>4=exp</UP>{[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,
<UP>Equivalents=2 × </UP>[(<UP>F1H+2 × F1H<SUB>2</SUB>+F2H+2 × F2H<SUB>2</SUB></UP>)<UP>/</UP><IT>f<SUB>t</SUB></IT>] (Eq. 12)
Where ft 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 for E1-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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of eNOS Subdomains-- 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 eNOSox and eNOSred resulted in a single band on SDS-PAGE with apparent molecular masses of 54 and 82 kDa, respectively (Fig. 1A) and exhibited immunoreactivity with polyclonal antibodies against eNOS (Fig. 1B).


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Fig. 1.   Homogeneity of purified eNOSox and eNOSred. Purified eNOSred (lane 2) and eNOSox (lane 3) after nickel-chelating column chromatography were analyzed by SDS-PAGE/Coomassie Blue staining (A) and Western blotting (B). Molecular weight markers are shown in lane 1. Samples of approximately 1 µg were used for each run.

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 delta -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 (22). 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).

                              
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Table I
Stoichiometry of the cofactors of purified eNOS subdomains (n = 7)

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.

Spectroscopic Characteristics of eNOSox-- 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 alpha /beta band at 518 nm, 15.4 mM-1cm-1, and a charge-transfer band at 645 nm, 5.8 mM-1cm-1. Treatment with L-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 alpha /beta 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 (9, 13, 17). 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 (9, 13, 17).


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Fig. 2.   UV-visible spectra of eNOSox (solid line) and its L-arginine complex (long dash), ferrous form (medium dash-dot), and ferrous CO complex (short dash). The spectra between 500 and 700 nm are enlarged in the inset. Spectra for a typical preparation were given. Some eNOSox preparations show a purity index, i.e. A280/A396 in the L-arginine complex, which is as reliable as 1.3.

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 (the gmin was only observable at somewhat lower temperature, ~ 4 K), and the low spin heme show conspicuous rhombic g 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 (32, 33). 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 (33).


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Fig. 3.   EPR spectra of eNOSox (A), L-arginine complex (B), and imidazole complex (C). The concentration for eNOSox was 25 µM in 50 mM HEPES, pH 7.4, with 10% glycerol, 0.1 M NaCl. L-Arginine was 100 mM and imidazole was 40 mM. EPR conditions were: microwave frequency, 9.61 gHz; power, 1 milliwatt; modulation, 10 G; temperature, 11 K; time constant, 0.33 s. Each spectrum is the average of two scans.

Stoichiometric Titration of the eNOSox-- To determine the redox capacity of the purified eNOSox, a stoichiometric titration was conducted using standardized dithionite solution. L-Arginine was added to make the heme homogeneously high spin. The spectral conversion from ferric to ferrous heme during the course of titration appeared to involve only one simple redox reaction as evidenced by the isosbestic points at 410, 489, 532, 615, and 678 nm for both the absolute and difference spectra relative to the resting eNOSox spectrum. An isosbestic point at 338 nm was slightly perturbed by dithionite whose absorption peaks at 314 nm (Fig. 4, A and inset). However, the optical amplitude changes at 444-388 nm showed a long lag for 1.5-2.5 (in three separate titrations) reducing equivalents before a sharp rise. 1-1.5 reducing equivalents were needed to completely reduce eNOSox (Fig. 4B). The additional 1.5-2.5 reducing equivalents consumed during titration were not due to oxygen contamination as assessed by lumiflavin titration using the same titration vessel and conditions. Furthermore, similar stoichiometric titrations performed on eNOSred did not show an initial long lag (see below). The extra reducing equivalents used to titrate eNOSox could be due to oxidized biopterin or free sulfhydryl group at the protein surface. The former may be a consequence of autoxidation of BH4, and the latter could be due to the loss of the zinc cluster, which coordinates with two cysteines from each monomer (2, 5, 6). However, the samples used in these titrations are not BH4-replenished. The content of biopterin was as low as 0.2-0.3/heme and was preset as BH2, which is not reducible by dithionite. This left the zinc loss as the most possible cause of the additional consumption of dithionite in the titration. Three experiments were conducted to assess this hypothesis. Zinc analysis by ICP-MS analysis was carried out using either eNOSox or purified whole eNOS. Sufficient amount of zinc was determined by ICP-MS analysis (data not shown). Gel filtration chromatography was conducted to determine the population of eNOSox monomer and dimer. Molecular sieving using five molecular weight standards and purified eNOSox indicated that the whole population was present as a dimer with a molecular mass of >100 kDa (Fig. 5). Titration of free thiol by 4,4'-dithiopyridine was also conducted on eNOSox using free L-cysteine as a positive control. Time-dependent modification of the thiol was monitored in parallel with urea-treated eNOSox and a bovine eNOSox predetermined to have zinc and present as a dimer (Fig. 6) (5). Both yeast-expressed human eNOSox and eNOSox, trypsinolyzed from bacterial expressed bovine eNOS, exhibited almost identical kinetics of chemical modification (Fig. 6). Two to three thiol groups were easily modified in both protein samples at a rate of 0.5 min-1, but the next six residues were modified much slower at 0.02 min-1. Pre-treatment of 5 M urea significantly enhanced the extent of chemical modification in the fast phase as a result of exposure of about additional two to three thiol groups. The rates of the two phases remain similar, 0.5 and 0.02 min-1, respectively, but the contribution of each phase shifted from 2:5 to 4:2.5 after urea treatment. Moreover, the overall extent of modification after a 2-h period remained the same as the eNOSox sample not treated by urea. In contrast, free L-cysteine followed simple modification kinetics, and the rate, 8 min-1, is even faster than the fast phase of that observed for eNOSox samples.


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Fig. 4.   Stoichiometric titration of eNOSox by sodium dithionite in the presence of L-arginine. Purified eNOSox, not replenished with exogenous BH4, at 18.0 µM and containing 500 µM L-arginine, was titrated by 16.2 mM sodium dithionite anaerobically. A 1.8-ml total volume of reaction mixture in 50 mM HEPES, pH 7.5, containing 0.1 M NaCl and 10% glycerol, was used in the titration. Panel A shows the absorption spectra during the reductive titration. The inset shows the difference spectra from panel A (against resting). Panel B gives the changes of A444 minus A388 as a function of reducing equivalent per mol of eNOSox. The solid straight lines indicate the initial, ending levels and the initial slope of the heme titration.


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Fig. 5.   Molecular mass estimation by gel filtration. eNOSox (~1 mg) was gel-filtered through a Sephacryl 200 HR column (1.5 × 50 cm), the ratios of sample elution volume (Ve) and column void volume (Vo) were determined by blue dextran and plotted with five molecular mass standards: horse heart cytochrome c (12,400 Da), bovine erythrocytes carbonic anhydrase (29,000 Da), bovine serum albumin (66,000 Da), yeast alcohol dehydrogenase (150,000 Da), and sweet potato beta -amylase (200,000 Da) (solid circles). The data for eNOSox (solid triangle) was interpolated into the standard curve to obtain the estimated molecular mass. There was only one protein peak monitored at A280 absorbance detected for eNOSox in the gel filtration profile.


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Fig. 6.   Kinetics of chemical modification for thiols by 4,4'-dithiopyridine. 50 µM 4,4'-dithiopyridine was added individually to 4 µM human eNOSox (solid circles), 4 µM bovine eNOSox (open circles), 4 µM human eNOSox pretreated with 5 M urea for 2 h (cross), and 9 µM free L-cysteine (open triangles) in 50 mM KPi, pH 7.5. Formation of 4-thiopyridine after reaction with the -SH groups of each sample was monitored at 324 nm (epsilon  = 19.8 mM-1cm-1) on an HP8453 diode-array spectrophotometer for a period of 2 h at room temperature. The solid lines are one- or two-exponential fits for each set of kinetic data.

Tetrahydrobiopterin Radical Formation of eNOSox-- BH4- reconstituted eNOSox prepared at a concentration of ~300 µM was premixed with excess L-arginine and reacted with oxygenated buffer anaerobically at room temperature and freeze-trapped at several time points. The EPR spectrum corresponding to a 100-ms reaction time is given in Fig. 7. EPR recorded at 11 K between 200 and 4200 G revealed both the heme component and the radical component (Fig. 7A). The EPR spectrum of a control L-arginine-treated eNOSox was also recorded under the same EPR conditions. Two spectra are normalized to the same concentration of heme. Approximately 50% of the BH4 was converted to the BH4 radical, and other diamagnetic heme intermediates were estimated from the decrease of the high spin heme signal amplitude. The radical signal observed at the g = 2 region was measured again at 115 K (Fig. 7B). The hyperfine features pertaining to nitrogen and proton splittings are clearly revealed. The biopterin radical was centered at g = 2.002 and had an overall line width of 39 G. Microwave power dependence indicated a strong magnetic interaction with the heme center with a P1/2 as high as 14 milliwatts at 120 K (data not shown). Spin concentration was estimated by double integration of the EPR signal, using a copper standard, to be ~20 µM. After correction for the ~4-fold dilution factor during rapid freezing, we essentially observed ~80 µM radical, equivalent to about 40% of the total biopterin because BH4/heme ratio was ~0.7. This radical EPR could be closely simulated by including one strongly coupled nitrogen at N5, one alpha proton at N5, and one beta proton at C6 (dashed line in Fig. 7B).


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Fig. 7.   Transient formation of BH4 radical by eNOSox. 280 µM eNOSox containing 0.7 equivalent of BH4/heme was first mixed with 1 mM L-arginine then reduced by dithionite titration in a tonometer with a side-arm attaching an optical cuvette. This ferrous eNOSox was reacted with oxygenated buffer at a 1:1 ratio on a rapid-freeze quench apparatus, the reaction mixture was freeze-quenched in isopentane, and the ice particles were collected at several reaction times from 20 to 200 ms at a ram velocity of 2.5 cm/s at room temperature by our specially designed packing device (42). Liquid helium EPR (A) was recorded for the intermediate trapped at 100 ms (solid) with a parallel control sample of L-arginine-treated eNOSox (dash). EPR conditions were the same as those in Fig. 3. Liquid nitrogen temperature EPR was recorded in the radical region in A as a 100-G scan. The EPR conditions were: microwave frequency: 9.29 gHz; power, 1 milliwatt; modulation, 2 G; time constant, 0.33 s; and temperature, 115 K. The spectrum in B (solid) was from a single scan. Dashed lines is a computer simulation using the following parameters: gx = gy = gz = 2.0023; line width, 12/11/11 G. The Axx/Ayy/Azz values: for the nitrogen nucleus, 2/1.5/23 G; for the two hydrogens, 4.6/21.6/11.8 G and 12.4/10.9/14.0 G, respectively.

Enzyme Activities of eNOSred-- Cytochrome c reductase activity, DCPIP, and ferricyanide reduction activities were evaluated for eNOSred expressed in yeast (Table II). Cytochrome c reductase activity was 70.3 mol/min/mol and was increased about 2- to 3-fold, 194.3 mol/min/mol, in the presence of Ca+2/CaM. This effect of CaM was less than we previously observed for baculovirus-expressed eNOSred (17). However, the absolute activity, both in the presence and absence of CaM, are higher than the values reported for sf9-expressed eNOSred and eNOS, i.e. 13.8/20.9 and 138/224 without and with Ca+2/CaM, respectively. Both ferricyanide and DCPIP reductase activities were greater than that of cytochrome c reductase. Ferricyanide reductase activity increased from 3220 to 4480 min-1 by Ca+2/CaM, whereas DCPIP reductase activity was increased from 400 to 800 min-1 by adding Ca+2/CaM (Table II). Our data are compatible with literature data for eNOSred or full eNOS (Table II). Although there are some variations of cytochrome c and ferricyanide reductase activities among different eNOS or eNOSred preparations, most of them are within the same order of magnitude (Table II). Differences in assay temperature, expression system, and buffer composition could be the reasons that resulted in these variations.

                              
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Table II
Catalytic activity of eNOSred and eNOS

Flavin Fluorescence of eNOSred and Its Interaction with CaM-- The emission spectrum of isolated eNOSred shows a broad band from 470 to 650 nm peaked at ~528 nm (Fig. 8). The intensity of the fluorescence was increased by ~30% in the presence of Ca+2/CaM, but there was no obvious shift of the peak. Excess EDTA could only reverse ~80% of the fluorescence change caused by Ca+2/CaM (Fig. 8). This residual fluorescence increase, which is not reversed by EDTA, could be simply the slight increase of intensity caused by Ca+2 alone (data not shown). This fluorescence change provided a nice index to determine the CaM binding. Titration of micromolar level of eNOSred with CaM in the presence of Ca+2 showed a sharp breaking point at 1:1 ratio of CaM to eNOSred (inset of Fig. 8). This result indicated that the Kd value of CaM is significantly lower than micromolar, and each reductase domain binds one CaM.


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Fig. 8.   Calmodulin effect on flavin fluorescence. Flavin emission spectra between 450 and 650 nm of 1.9 µM eNOSred were recorded before (solid line) and after addition of 250 µM Ca2+ and 3.4 µM calmodulin (long dash) to follow the formation of the CaM·eNOSred complex and the dissociation of this complex by 3.5 mM EDTA addition (short dash). A negative control, excluding eNOSred and CaM was subtracted from the data. Inset, stoichiometric titration of eNOSred by CaM monitored by flavin fluorescence change at 530 nm. The intersection of two straight lines is CaM binding stoichiometry.

Stoichiometric Titration of eNOSred-- Identical anaerobic titration procedure as eNOSox titration was applied here to determine the redox capacity in eNOSred using dithionite as titrant. A total of four reducing equivalents, required to fully reduce eNOSred, accounted exactly for the capacity of two flavin cofactors (Fig. 9). No additional redox centers other than the FAD and FMN were disclosed by this titration. There are three stages of reduction observed in the dithionite titration. The first stage took one reducing equivalent, and the electronic spectra showed isosbestic points (~366 and ~508 nm) in this process (Fig. 9A), indicating a single redox transformation step. The absorbance decrease at 456 nm, and the corresponding increase at 600 nm are attributed to the formation of a neutral flavin semiquinone. The second stage took two reducing equivalents. It appeared to show one isosbestic point (~342 nm) but is not conclusive, indicating that this stage is likely to be involved in at least two chemical reaction steps (Fig. 9B). There was a large decrease of 456-nm absorbance accompanied by a small change at 600 nm. The last stage involved one-electron reduction to reach the fully reduced state (Fig. 9C). The further bleaching of 456- and 600-nm absorbance evidenced disappearance of both the oxidized flavins and flavin semiquinone. The general titration profile is very similar to that published for microsomal CPR and nNOS reductase domain (43, 44).


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Fig. 9.   Stoichiometric titration of eNOSred and computer simulations. eNOSred at 65 µM in 50 mM HEPES, pH 7.5, containing 0.1 M NaCl and 10% glycerol, was titrated with standardized 30.8 mM sodium dithionite anaerobically. Panel A shows the absorption spectra (1-5) up to the addition of 1 reducing equivalent. The spectra of the fully oxidized flavins (dash-dot-dash) and semiquinone form of FMN (solid line) are highlighted. Panel B shows the spectra 5-10 for the titration between 1 and 3 reducing equivalents, whereas the spectra 10-20 shown in panel C represent the titration data for addition of the 3-6 reducing equivalents. The hydroquinone of the second flavin (FADH2) (solid line) and FADH· semiquinone (dash-dot-dash) are highlighted in C. Panel D is the plot of absorbance changes at 456, 508, and 600 nm against the reducing equivalents consumed per mol of eNOS red. Lines going through data points of each wavelength are the simulation obtained as detailed in the main text. The arrows in A-C indicate the direction of the spectra changes with increasing dithionite. Another two duplications of titration show very similar results.

Computer Simulations for the Reductive Titration Data-- The data shown in Fig. 9D at three different wavelengths were simulated by the SCoP program according to Equations 1-12 to obtain three redox potentials gaps, Delta E1-Delta E3, between four half-reactions of two flavins in eNOSred (31). Computer simulation for the data at 456, 508, and 600 nm was successful as indicated by the close match of the simulations and the actual data except the initial <0.3 reducing equivalents (Fig. 9D). The initial short lag was probably due to the residual amount of oxygen in the titrator. This simulation process was tested using any arbitrary absolute midpoint potential value for one of the four half-reactions and to zoom in the values for Delta E1-Delta E3. The variation for each of the redox potential gaps is not significant as shown in Table III. The optimal value for Delta E1 is the largest, 180 mV, and a clean conversion from one oxidized flavin to the semiquinone form was expected in the first stage of titration. In contrast, Delta E2 was almost zero, indicating that the second stage of reduction consisted of two almost parallel half-reactions. The value of Delta E3, 73 mV, was in the middle and could be used to estimate the cutoff point for obtaining the absorbance contribution from only one specific half-reaction. The extinction coefficients at three different wavelengths were also converged by several simulation cycles and are shown in Table IV. In principal, we could conduct these simulation cycles on any wavelength between 300 and 700 nm and reconstruct the spectrum for each of the six flavin redox species.

                              
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Table III
The three redox potential gaps of the four half-reactions of flavins of eNOSred and related redox systems

                              
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Table IV
Extinction coefficients of the different redox states of flavins in eNOSred at different wavelengths


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have successfully prepared both eNOSox and eNOSred using the yeast expression system. Purified eNOSox domains appear to have most of the heme characteristics in intact eNOS. The optical spectra of ferric and ferrous eNOSox, their ferrous-CO complex, and the spin state change caused by L-arginine are all typical for eNOS and eNOSox as we observed previously (17, 21, 24). The purity index, expressed as the ratio of A280/A396, was as reliable as 1.3-1.6 compared with 1.5-1.7 found in our previous baculovirus-expressed recombinant protein (17). This ratio is not as reliable as the recent iNOSox preparation expressed from E. coli (9), but our oxygenase domain does not have the N-terminal heterogeneity as observed for iNOSox as only one single peptide band was observed on SDS-PAGE at the ~50-kDa region (9).

The EPR spectrum of the isolated eNOSox showed a dominant low spin P450-type heme. Using the low field g = 7.5 signal amplitude to estimate the spin state population against the L-arginine-treated eNOSox, about 75% of the heme was present as the low spin form. This high proportion of low spin heme is at odds with the room temperature optical data (Fig. 2 versus Fig. 3) judging from the similar amplitude of the charge-transfer band at 650 nm of the resting eNOSox and eNOSox treated with L-arginine. Optical data implied that the majority of the heme was present as the high spin form, thus a temperature-dependent heme spin state change likely occurred with the low spin heme electronic configuration as the ground state. A Truth Diagram analysis for the correlation between heme rhombicity and axial ligand field strength for the low spin heme component put the eNOSox at the lower right edge of the P zone, indicating a P450-like protein (33). The rhombic distortion (V), axial perturbation (Delta ), and heme rhombicity (%) obtained were 2.09, 5.75, and 36.4, respectively. There might be another low spin component present as indicated by the shoulder at the g = 2.43 signal. However, this additional component was not as visible as that observed for full eNOS, and only the low rhombicity low spin heme component appeared to be present in the isolated eNOSox (33). In contrast, the imidazole low spin heme complex showed two better-resolved EPR species than the corresponding imidazole complex of full eNOS (compare Fig. 3, spectrum C, with Fig. 6a in Ref. 33) with very similar g values for the two sets of low spin heme complexes. The calculated values of V, Delta , and percent rhombicity for the two low spin species are 2.52/4.53/56% and 2.63/4.05/65%, respectively, and they place these two heme complexes at the center of the P zone. Throughout the entire low spin imidazole·heme complex, there was a reciprocal relationship between the heme rhombicity and the tetragonality, indicating that the heme rhombic distortion attenuates the axial ligand intensity, possibly via a lengthening of the Fe-S bond.

The extra 1.5-2.5 reducing equivalents required to initiate the heme reduction in the stoichiometric titration is puzzling (Fig. 4). These additional equivalents did not originate from biopterin, because the amount of pterin was too low to account for this amount of reducing equivalents and dithionite does not reduce BH2 to BH4 due to unfavorable redox thermodynamics (35). The zinc loss is not the reason: there was plenty of zinc present in isolated eNOSox as assessed by ICP-MS analysis. The isolated eNOSox is a perfect dimer as analyzed by gel filtration. The monomeric form was not even detected. Furthermore, the titration for the surface-exposed thiol function group showed identical modification kinetics as another bovine eNOSox whose crystallographic data indicate the presence of zinc cluster. Thus the hypothesis that a zinc loss leading to surface-exposed disulfide linkage as that found in the iNOSox crystallographic data does not apply to our isolated eNOSox (7). Furthermore, the possibility of propensity of zinc loss in recombinant eNOSox but not in intact eNOS may be unfounded in our yeast expression system. We do not see additional metal redox centers such as heme, iron-sulfur cluster, or copper by optical or EPR spectroscopy, thus leaving us with no explanation for the extra reducing equivalents that show much higher redox potential than the heme center.

Reduction of the heme center appeared to consume more than one reducing equivalent (1-1.5 in three experiments). A similar case in iNOSox was also observed recently (9). The sharp increment of absorbance change at the beginning of the titration and a curvature and even tailing approaching the end of the titration seem to indicate that the titrant may not have electron-donating power strong enough to completely reduce all of the heme molecules. Considering the very negative midpoint potential of thiolate-ligated heme, it is possible in the later portion of the titration that only part of the reducing equivalents from dithionite were donated to the heme center dictated by the midpoint potential difference between the heme and dithionite (36). We thus put more emphasis in using the initial linear sharp rise to estimate the end point of titration. By doing this, we get a stoichiometry closer to 1 rather than 1.5.

The biological activity of our eNOSox was demonstrated by its capability in forming the biopterin radical (Fig. 7). We chose this method because it is directly linked to the redox function of the protein and provides detailed information regarding the reaction mechanism of eNOS. In our study, the radical signal plateaued at ~100 ms at room temperature. The line shape, intensity, and initial kinetics of biopterin radical formation appeared very similar to those published for iNOSox (26, 27). Computer simulation for the BH4 radical indicates a minimal requirement for one nitrogen and two proton nuclei to match the EPR data. Because N5 (or its 4a carbon) is positioned para to the electron-releasing amino group at C2 and thus has high electronegativity, it is thus more favorable than N8 to give the first electron. N8 (or its 8a carbon) is positioned meta to C2 and C4; thus electron withdrawal can only occur via conjugation with 4-oxo group. The pKa of the N5 proton is much higher than neutral and is not dissociated easily; thus a hyperfine interaction of this proton with the unpaired electron at N5 is expected. The second proton has to come from the C6 beta proton. Such initial trial of simulation appears fairly promising. Our observed biopterin radical is thus likely a BH<UP><SUB>4</SUB><SUP>+</SUP></UP>· cation radical (37, 38). Although N8 nuclei and its associated proton have been proposed to be involve in the unpaired spin system (37), it remains to be clarified by further spectroscopic studies using isotope replacement. Nonetheless, we present here the first high quality EPR spectrum of eNOS biopterin radical and will pursue the mechanistic role of biopterin using a rapid-freeze EPR approach in parallel with stopped-flow and other kinetic methods.

eNOSred activity was assessed by three different assays. The cytochrome c reduction and the DCPIP reduction assays require the participation from both flavins, and ferricyanide reduction activities were believed to involve only FAD (39). In all cases, CaM enhanced the activity and the enhancement for cytochrome c reduction and DCPIP reduction activity to a similar extent. Why we only see a ~3-fold activity increase for the cytochrome c reductase activity by CaM using our yeast protein and a 10-fold increase in our previous sf9-expressed eNOSred is unclear (Table II). Nonetheless, CaM appeared to interact both between FMN and the heme as well as between FAD and FMN as initially observed in nNOS (40). There are many factors that enhance the reductase activity of eNOS, including the CaM binding, the removal of the autoinhibitory peptide, and the phosphorylation of the C terminus of the reductase (1, 39, 41, 42). Furthermore, the presence of dithiothreitol, EDTA, and variation in ionic strength during different stages of the purification also affect the sensitivity of the reductase domain activity to Ca+2/CaM (17, 39, 41, 42). Comparing eNOSred activity values determined from different laboratories does not yield straightforward conclusions. The effect of CaM on nNOSred does not appear to shift the midpoint potential of either half-reaction of the two flavins (43), because addition of CaM caused only marginal change for the FMN/FMNH midpoint potential and essentially no change for the other three half-reactions.

The only redox centers present in eNOSred are the two flavins, because exactly 4 reducing equivalents were consumed in the stoichiometric titration (Fig. 9D). Optical changes of the flavins occurring almost immediately after dithionite addition contrasts with the data for eNOSox, which required 1.5-2.5 additional reducing equivalents before reduction of heme and supports that additional redox centers were present in eNOSox (Fig. 4B). In addition to quantifying the redox capacity of eNOSred, stoichiometric titration also enabled determination of the relative redox potentials between different half-reactions as illustrated in this study (Fig. 9D and Table III). Successful simulation in the data for all three wavelengths, using the same set of difference midpoint potential values, attested to the utility of this approach. The redox potential gap between the first and second half-reaction was 170-190 mV, thus a complete separation of the first half-reaction from the other three is expected. The optical spectrum at the point of addition of one reducing equivalent should contain one intact flavin (FAD) and one flavin semiquinone (FMNH·) (Fig. 9A, heavy line). The extinction coefficient for the FMNH· semiquinone could thus be unambiguously determined. The spectral change at 600 nm is completely due to semiquinone forms of the flavins, because the fully reduced and fully oxidized samples were silent in this region. The titration data indicate that the second semiquinone was gradually reduced to its hydroquinone at the addition of the fourth reducing equivalent. However, Delta E3 was only 56-90 mV and prohibited clean separation of the last half-reaction from the other three. Because 10Delta E3/0.059 = (Ox1 · Red2)/(Ox2 · Red1) for the last two overlapping half-reactions,
<UP>Ox</UP><SUB>1</SUB>+<UP>e</UP><SUP>−</SUP>=<UP>Red</UP><SUB>1</SUB>

<UP><SC>Reaction</SC> 1</UP>

<UP>Ox</UP><SUB>2</SUB>+<UP>e</UP><SUP>−</SUP>=<UP>Red</UP><SUB>2</SUB>

<UP><SC>Reaction</SC> 2</UP>
we expect that only 70-80% of the reaction after addition of the fourth electron is only contributed by FADH· semiquinone reduction to FADH2. Simulation for the 600-nm data here was very useful to define the extinction coefficient for the second semiquinone species, because the trapezoidal titration profile is very sensitive to the difference midpoint potential as well as the extinction coefficient (31). Thus, simulation greatly assisted in converging the value of the Delta E values and the extinction for the second flavin semiquinone. There was a 20% difference in the extinction coefficient at 600 nm for these two flavin semiquinones and almost a 3-fold difference at 456 nm and a difference of ~50% at 508 nm with the FMNH· having the higher values (Table IV). The middle two half-reactions, corresponding to that after addition of the second and third reducing equivalents, attributable to the formation of FAD semiquinone and FMNH· transformation into the fully reduced form, were almost equivalent in redox potential and were titrated together. The absolute values of the midpoint potential for all four half-reactions will be determined by potentiometric titration. Once these values are available, they will be used to validate the Delta E values obtained in this study and to refine the accuracy of the extinction coefficient derived herein. The spectral contribution from each half-reaction at every single wavelength can be reliably determined and will be useful in future mechanistic studies using stopped-flow measurements.

Comparing the Delta E values with other diflavin reductase proteins (Table III), eNOSred resembles closely the rabbit P450 reductase in all three potential gaps. Our results differ from those for nNOSred (Delta E1 was 40 mV larger and Delta E2 was 40 mV smaller), P450BM3 (Delta E1 was very small and Delta E2 was 80 mV larger), and sulfite reductase (Delta E2 was 55 mV larger and Delta E3 was 130 mV smaller). These differences have a major impact on the thermodynamic control of electron transfer and the nature of the half-reaction that couples with the heme reduction (31, 43-45).

    ACKNOWLEDGEMENTS

We thank Dr. Susan Cates at Rice University, Department of Biochemistry and Cell Biology, for initial efforts in measuring the eNOSox molecular weight using equilibrium centrifugations. We also thank Dr. Robert E. Serfass at the University of Texas Medical Branch at Galveston, Department of Preventive Medicine and Community Health, who performed ICP mass analysis for zinc in our eNOSox and eNOS proteins. A bovine eNOSox sample, given by Dr. C. S. Raman, University of Texas Health Science Center, Department of Biochemistry and Molecular Biology, for our chemical modification studies is very much appreciated.

    FOOTNOTES

* This work was supported by United States Public Health Services Grant GM56818 (to A.-L. T.) and Grant HL60625 (to L.-H. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed almost equally to this work.

§ To whom correspondence should be addressed: Division of Hematology, University of Texas Health Science Center, P. O. Box 20708, Houston, TX 77225. Tel.: 713-500-6771; Fax: 713-500-6810; E-mail: Ah-lim.Tsai@uth.tmc.edu.

Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M209606200

    ABBREVIATIONS

The 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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