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Originally published In Press as doi:10.1074/jbc.M704890200 on August 13, 2007

J. Biol. Chem., Vol. 282, Issue 39, 28557-28565, September 28, 2007
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Oxygen Metabolism by Endothelial Nitric-oxide Synthase*

Ying Tong Gao{ddagger}, Linda J. Roman{ddagger}, Pavel Martásek§, Satya Prakash Panda{ddagger}, Yuzuru Ishimura, and Bettie Sue S. Masters{ddagger}1

From the {ddagger}Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, the §Department of Pediatrics, Charles University School of Medicine I, 128 08 Prague, Czech Republic, and the Department of Biochemistry and Integrated Biology, School of Medicine, Keio University, Tokyo 160-8582, Japan

Received for publication, June 13, 2007 , and in revised form, August 10, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric-oxide synthase (NOS) catalyzes both coupled and uncoupled reactions that generate nitric oxide and reactive oxygen species. Oxygen is often the overlooked substrate, and the oxygen metabolism catalyzed by NOS has been poorly defined. In this paper we focus on the oxygen stoichiometry and effects of substrate/cofactor binding on the endothelial NOS isoform (eNOS). In the presence of both L-arginine and tetrahydrobiopterin, eNOS is highly coupled (>90%), and the measured stoichiometry of O2/NADPH is very close to the theoretical value. We report for the first time that the presence of L-arginine stimulates oxygen uptake by eNOS. The fact that nonhydrolyzable L-arginine analogs are not stimulatory indicates that the occurrence of the coupled reaction, rather than the accelerated uncoupled reaction, is responsible for the L-arginine-dependent stimulation. The presence of 5,6,7,8-tetrahydrobiopterin quenched the uncoupled reactions and resulted in much less reactive oxygen species formation, whereas the presence of redox-incompetent 7,8-dihydrobiopterin demonstrates little quenching effect. These results reveal different mechanisms for oxygen metabolism for eNOS as opposed to nNOS and, perhaps, partially explain their functional differences.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric-oxide synthases (NOS)2 catalyze the formation of NO from L-arginine in two consecutive monooxygenation reactions, which include N{omega}-hydroxy-L-arginine (NOHA) as a tightly bound intermediate (1-3).

Formula 1REACTION 1

Formula 2REACTION 2

Formula 3REACTION 3

In addition to the reactions listed above, NOS also catalyzes the formation of reactive oxygen species (ROS) such as superoxide anion (Formula 3) and H2O2 (4-6). Reaction 1 is the first step, Reaction 2 is the second step, and Reaction 3 shows the overall reaction.

Formula 4REACTION 4
and/or

Formula 5REACTION 5

Unlike Reaction 3, where activated molecular oxygen is split to oxidize L-arginine to citrulline, NO, and H2O, Reactions 4 and 5 do not require the cleavage of the O-O bond. NOS is said to be "coupled" when all the electrons from NADPH are utilized in the formation of monooxygenated products and H2O (Reactions 1, 2, 3) and "uncoupled" when electrons leak to molecular oxygen to form ROS (Reactions 4 and 5).

Spontaneous dismutation (Reaction 6) of Formula 5 to H2O2 (k = ~2 x 105 M-1 s-1 at pH 7.4) produces H2O2 at a detectable level whether or not the NOS-catalyzed two-electron reduction of oxygen to form H2O2 as a direct product occurs.

Formula 6REACTION 6

Diacetyldeuteroheme-substituted horseradish peroxidase (dHRP) is an artificial trapping agent first used by Ishimura and co-workers (7, 8) to distinguish superoxide and hydrogen peroxide formation. dHRP rapidly (k > 106 M-1 s-1) reacts with Formula 6 and H2O2 to form compound III and compound II, respectively, according the equations below,

Formula 7REACTION 7

Formula 7REACTION 8
thereby effectively competing with Reaction 6 for the spontaneous mutation of Formula 7 to H2O2.

Compounds II and III are chemically stable and spectrally distinguishable from each other; thus, both Formula 7 and H2O2 can be trapped, and the initial product(s) of the uncoupled reaction(s) can be determined. We have modified the original dHRP trapping protocol for NADPH-consuming enzymes such as NOS and reported the successful combination of the dHRP trapping system with an NADPH regeneration system (9). Maintaining a constant low concentration of NADPH not only keeps NADPH from being oxidized by dHRP compound I but also keeps NADP+ "feedback inhibition" negligible. In this paper, we applied this modified dHRP trapping protocol to study the products of the eNOS reactions, both qualitatively and quantitatively.

Even though Reactions 4 and 5 appear to be simple chemical reactions, quantitative analysis has always been a challenge given the spontaneous/catalyzed dismutation of Formula 7. EPR is the logical analytical tool for studying the formation of small radicals such as Formula 7; however, quantification of Formula 7 to determine stoichiometry and determination of rates is difficult or impossible using EPR (5, 10). Our modified dHRP trapping protocol has been shown to be compatible with simultaneous measurement of oxygen concentration (9); in this paper we report the simultaneous measurement of oxygen consumption and dHRP compound III formation, revealing the uncoupled stoichiometry of eNOS.

Both NO and ROS are highly reactive chemical entities in aqueous solution; the complexity of their coexistence in a reaction mixture makes it difficult to determine stoichiometry (11-13). To simplify the system, superoxide dismutase (SOD) and catalase can be added to suppress accumulation of ROS and recycle partially reduced oxygen to either H2O or O2. The presence of SOD accelerates Reaction 6 to a rate that is diffusion-limited (14), and catalase catalyzes the breakdown of H2O2 to H2O and O2 (15).

Formula 9REACTION 9

In a system that contains sufficient amounts of both SOD and catalase, oxygen in the uncoupled reaction is fully reduced to water. Regardless of whether Formula 9 and/or H2O2 is the initial product, the overall uncoupled reaction will be as follows.

Formula 10REACTION 10

Considering all of the reactions listed above, it is apparent that molecular oxygen is the ultimate oxidizing reagent in both the coupled and uncoupled reactions by NOS; NADPH, on the other hand, is the ultimate electron donor that initiates all subsequent redox processes. The ratio of [O2]/[NADPH] varies in accordance with substrate/cofactor binding and serves as an important parameter to study coupling/uncoupling of NOS.

The fully coupled NOS reaction with L-arginine as substrate produces the theoretical value of [O2]/[NADPH] = 2/1.5 = 1.33 (Reaction 3). When NOHA is added as the substrate, the theoretical value becomes [O2]/[NADPH] = 2.0 (Reaction 2). In the absence of L-arginine and NOHA, NOS is totally uncoupled, and the theoretical value becomes [O2]/[NADPH] = 1/2 = 0.5 (Reaction 10). In the absence of SOD and catalase, we calculate the theoretical value of [O2]/[Formula 10] to be 1/1, when Formula 10 is the initial product of the uncoupled reaction (Reaction 4).

Tetrahydrobiopterin (BH4) is an essential cofactor in the oxygenase domain and is proposed to have multiple roles in all three mammalian NOS isoforms (16-18). One of the presently accepted functions of BH4 is to act as a one-electron donor during reductive activation of the oxyferrous complex of the heme (18-21). Although there are many publications about the roles of BH4 in eNOS and how the absence of BH4 promotes eNOS uncoupling (22-24), there is little quantitative evidence regarding its effect on oxygen metabolism. In this study, we report a decreased oxygen consumption rate, as well as a lower [O2]/[NADPH] ratio when BH4 is absent from the fully coupled eNOS reaction.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—NADPH was purchased from Alexis (San Diego, CA). 5,6,7,8-Tetrahydrobiopterin and 7,8-dihydrobiopterin (BH2) were from Research Biochemicals International (Natick, MA). Fe(III) 2,4-diacetyl deuteroporphyrin IX chloride was purchased from Porphyrin (now Frontier Scientific, Inc. Logan, UT). Trizma base, L-arginine, cytochrome c (horse heart), K3Fe(CN)6, DL-isocitric acid trisodium salt, L-arginine, and other commercial chemicals were obtained from Sigma-Aldrich.

Enzymes—eNOS was expressed in Escherichia coli and purified as described by Martásek et al. (25). The enzyme preparation was concentrated to 50 µM after being purified by a 2'5'-ADP-Sepharose 4B column and then dialyzed against 50 mM Tris-HCl (pH 7.4, with 100 mM NaCl and 5% glycerol) to remove 2'-AMP, arginine and BH4. NO Formation rate (hemoglobin capture assay) of the purified eNOS was determined to be 120 ± 10 nmol/min/mg, using the extinction coefficient of 60 mM-1 cm-1 at 401 nm.

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDP-2) was prepared as described by Cunningham and Bash (26), and Ding and co-workers (27) with help from Dr. Lee McAlister-Henn's lab (The University of Texas Health Science Center at San Antonio). The His-tagged protein was purified from a nickel column and concentrated to 8 mg/ml. Activity was tested by monitoring NADPH formation at 340 nm. Purified IDP-2 has stoichiometrically bound NADP+. The activity of the IDP-2, measured by monitoring the formation of NADPH at 340 nm with an extinction coefficient of 6220 M-1 cm-1, was calculated to be 40 µmol/min/mg.

dHRP was prepared using the method described by Makino et al. (7, 28). Native horseradish peroxidase was bought from Toyobo Inc. (Japan). Calmodulin was prepared by the method of Zhang and Vogel (29); SOD and catalase were purchased from Sigma-Aldrich.

dHRP Experiments—dHRP trapping of superoxide anion and hydrogen peroxide was monitored by an Agilent 8453 diode array UV-visible spectrophotometer. The standard reaction buffer consists of 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 200 µM CaCl2, and 50 µM diethylene triamine pentaacetic acid. The reaction was carried out in a glass cuvette, and measurements were taken every 10 s. A typical reaction mixture contained the standard reaction buffer and 100 µM dHRP, 200 µM MgCl2, 0.65 µM eNOS (BH4- and arginine-free), 2 µM IDP-2, and 10 mM DL-isocitric acid (trisodium salt) was added to start the reaction immediately prior to the first optical measurement. Where needed, calmodulin (7 µM) and SOD (23 µg/ml) were added as described in the figure legends. Control experiments were performed in the absence of eNOS.

Quantitative analysis of the uncoupled [O2]/[Formula 10] ratio was performed using simultaneous measurements of oxygen consumption and the abovementioned dHRP trapping of superoxide anion. The reaction was carried out in an air-tight cuvette (2.22 ml), and the oxygenated standard reaction buffer was premixed with 160 or 100 µM dHRP, 200 µM MgCl2, 2.3 µM eNOS (BH4- and arginine-free), and 2 µM IDP-2; 10 mM DL-isocitric acid was added to start the reaction. The rate of compound III accumulation was calculated using the 583-nm delta extinction coefficient of 3.8 mM-1 reported by Makino and Yamazaki (28), and the corresponding oxygen consumption was recorded by the oxygen sensing system described below.

Oxygen Consumption—The uptake of molecular oxygen was measured at 24 °C using a fluorescence-based oxygen sensing system consisting of an SD2000 dual channel spectrophotometer combined with an LS-450 Blue LED light source and a Foxy-18G probe, all from Ocean Optics (Dunedin, FL). The reaction was carried out in an air-tight cuvette (1.80 ml), designed and fabricated in the laboratory. The standard reaction buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 200 µM CaCl2, and 50 µM diethylene triamine pentaacetic acid) was stirred gently overnight to ensure full oxygenation. A typical reaction mixture contained 7 µM calmodulin, 23 µg/ml SOD, 1.7 µM catalase, 5.5 µM BH4, and 0.3 ~ 2 µM eNOS. To study the coupled reaction, either 500 µM L-arginine or NOHA may be added. The reaction was started by the addition of NADPH to a final concentration of 440 µM.

Determination of the [O2]/[NADPH] ratio was performed by injecting a small volume of NADPH quantitatively and monitoring the oxygen concentration trace. The reaction was carried out in the abovementioned oxygen apparatus. Standard reaction buffer containing 7 µM calmodulin, 23 µg/ml SOD, 1.7 µM catalase, 5.5 µM BH4, and 0.3-0.5 µM eNOS was mixed before multiple stepwise injections of NADPH, which started the reaction. Each injection contained a precisely determined amount of NADPH and could only reduce a certain amount of oxygen. The [O2]/[NADPH] ratio was determined under three conditions: 1) in the totally uncoupled reaction in the absence of L-arginine or NOHA, 2) in the presence of 500 µM L-arginine as substrate, and 3) in the presence of 500 µM NOHA as substrate.

It is noteworthy that excessive amounts of SOD and catalase were present in the oxygen consumption experiments to avoid accumulation of ROS. Each experiment contains multiple injections of NADPH, and the results were averaged. The resulting O2 uptake at each addition was within ±3%. At the end of each experiment, an excessive amount of sodium dithionite was added to ensure depletion of oxygen. At least two different preparations of eNOS were used to confirm stoichiometry in each instance.


Figure 1
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  		FIGURE 1.
Qualitative analysis of eNOS uncoupled reactions and the CaM effect. The reaction mixture contained 400 µl of reaction buffer, 100 µM dHRP, 200 µM MgCl2, 5 µM eNOS (BH4- and arginine-free), 2 µM IDP-2, and 10 mM DL-isocitric acid. A, dHRP compound III formation in the presence of 7 µM CaM introduced to the reaction mixture before the reaction started; optical measurements were taken every 10 s for 2 min. B, CaM effect: a plot of 583-nm absorption versus time in the presence and absence of 7 µM CaM.

 
In the absence of L-arginine or NOHA, the reaction was totally uncoupled. When either L-arginine or NOHA was added, eNOS was partially uncoupled (more in the discussion section), and the overall oxygen consumption equals the weighted sum of both the coupled and uncoupled reactions. The percentage of coupling is calculated by comparing the theoretical values of [O2]/[NADPH] to the experimental values.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Uncoupled Reaction and the Effect of CaM—The basic uncoupled reaction, performed in the presence of dHRP, CaM, and eNOS, free of BH4 and L-arginine, showed the formation of compound III of dHRP (Fig. 1A), indicating Formula 10 formation by eNOS. Sharp isosbestic points at 481, 530, and 605 nm were observed, indicating that the transition from ferric dHRP ({lambda}max = 507 and 640 nm) to dHRP compound III is the only reaction occurring, and that Formula 10 was the only product formed (~4 min-1) under such conditions. The control experiment, performed in the absence of eNOS, showed no spectral changes over 10 min.

The abovementioned reaction was then repeated in the absence of CaM. Exactly the same optical changes were observed (spectra not shown) but at much slower rate (~1 min-1). A plot of spectral changes at 583 nm is shown in Fig. 1B; in the first 2 min, the reaction rates in the presence and absence of CaM were both linear, but the (+)CaM rate is about three to four times faster than the (-)CaM rate. Our results are consistent with the CaM effect reported by Vásquez-Vivár et al. (30) using EPR spectroscopy.

Quantitative analysis of the uncoupled reaction was performed by simultaneous measurement of oxygen consumption and Formula 10 formation (9), and the uncoupled [O2]/[Formula 10] stoichiometry was obtained by comparing the rate of oxygen consumption to the rate of Formula 10 formation. Although the oxygen concentration was continuously monitored by the oxygen sensing system described under "Experimental Procedures," a diode array UV-visible spectrophotometer recorded a series of spectra of the reaction mixture every 10 s. When a spectrum was recorded, the burst of light from the diode array spectrophotometer interfered with the oxygen sensor, leaving ticks visible on the oxygen concentration trace at 10-s intervals (Fig. 2A); time alignment was achieved using these small ticks as an internal reference.

Difference spectra of dHRP compound III were produced by subtracting the spectrum of ferric dHRP from those recorded every 10 s after the reaction started (Fig. 2B). The sharp isosbestic points in Fig. 2B confirm that compound III formation (Reaction 7) is the only reaction occurring during the initial 2-min time course. The oxygen consumption trace, recorded simultaneously on the same sample, is shown in Fig. 2A. The rates of both Formula 10 formation and O2 consumption were calculated to be 5.4 µM/min, reflecting a stoichiometry of [O2]/[Formula 10] = 1/1 (Reaction 4).

The Arginine Effect—L-Arginine was found to stimulate the oxygen consumption rate by eNOS ~10-fold, depending on the enzyme preparation. This is a surprising finding given our previous publication (9) indicating that the oxygen consumption rate by nNOS is strongly inhibited by L-arginine. A comparison is shown in Fig. 3A, in which the oxygen consumption rate by eNOS was stimulated from ~4 to ~40 min-1, whereas the oxygen consumption rate by nNOS was inhibited from ~500 to ~140 min-1.

In contrast, nonhydrolyzable L-arginine analogs, such as N{omega}-nitro-L-arginine methylester, NG-monomethyl L-arginine citrate, and L-citrulline (the product of Reaction 3) did not stimulate oxygen consumption by eNOS (Fig. 3, B and C). The significance of these experiments and the comparison of the L-arginine effect on eNOS and nNOS will be addressed in detail under "Discussion."

The Tetrahydrobiopterin Effect—BH4 stimulates the oxygen consumption rate of eNOS by ~3-fold in the presence of L-arginine (Fig. 4A) but has no effect on oxygen consumption in the absence of L-arginine (data not shown). To evaluate the amount of ROS generated in a reaction mixture, SOD and catalase were introduced sequentially to the reaction mixture, whereas oxygen concentration was monitored. For example, the reaction could start in the presence of SOD, which keeps ROS in the form of H2O2 (Reaction 6); after ~100 µM of oxygen was consumed, catalase could be introduced to the reaction mixture, and H2O2 would be rapidly converted to molecular oxygen (Reaction 9), which can be monitored/recorded by the oxygen sensing system. BH4 almost totally inhibited ROS formation by eNOS, but ROS formation occurs in the absence of BH4 or the presence of BH2, a redox-incompetent BH4 analog (Fig. 4, B and C).


Figure 2
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  		FIGURE 2.
Simultaneous measurement of oxygen consumption and dHRP compound III formation. The reaction mixture contained oxygenated reaction buffer, 100 µM dHRP, 200 µM MgCl2, 1.2 µM eNOS (BH4- and arginine-free), 2 µM IDP-2, and 10 mM DL-isocitric acid. A, oxygen concentration trace. B, difference spectra of compound III accumulation every 10 s. The solid arrow indicates the elapse of time, and empty arrows indicate isosbestic points.

 
Fig. 4B indicates that a significant amount of ROS was formed by BH4-free eNOS even in the presence of L-arginine (detailed conditions are indicated in the figure legend). Upon the addition of catalase, the evolution of oxygen was observed to be 20-30 µM. Repeating the experiment with BH2-bound eNOS produced a similar result (Fig. 4C), but repeating the experiment with BH4-replete eNOS yielded a negligible amount of oxygen evolution (Fig. 4D). Although these experiments are not strictly quantitative, they present a qualitative measure of ROS formation under different conditions.

Because BH4 itself is a strong reducing reagent and may react with ROS directly, the experiment presented in Fig. 4D was repeated with a range of BH4 concentrations (500 µM to 10 mM). The same result was obtained (data not shown), indicating that the diminished formation of ROS by BH4-replete eNOS, rather than the ROS scavenging activity by BH4 itself, is responsible for the striking differences in the comparison of Fig. 4 (compare B and C with D). The literature indicates that free BH4 and eNOS-bound BH4 degrade at neutral pH at 0.017 and 0.006 min-1, respectively (31), rates that are insufficient to compete with eNOS-catalyzed oxygen consumption.


Figure 3
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  		FIGURE 3.
L-Arginine effect of oxygen uptake. The nNOS trace shown in this picture was taken from our previous publication for comparison (9). Oxygenated standard reaction buffer and 7 µM CaM, 23 µg/ml SOD, 1.7 µM catalase, 5.5 µM BH4, and 1.2 µM eNOS were mixed before the addition of 440 µM NADPH. A, L-arginine effect (500 µM L-arginine after addition). B, N{omega}-nitro-L-arginine methylester (L-NAME) and NG-monomethyl L-arginine citrate (L-NMMA) were introduced after the reaction started; no stimulation was observed. C, two additions of L-citrulline were introduced after the reaction started; no stimulation was observed.

 
Stoichiometry and eNOS Coupling—The determination of the [O2]/[NADPH] ratio is shown in Fig. 5. In the absence of L-arginine or NOHA, the reaction was fully uncoupled, and the measured [O2]/[NADPH] ratio was 0.50, the same as the theoretical value (Table 1). The same ratio measured with L-arginine or NOHA was slightly lower than the theoretical value (Table 1), indicating that eNOS was highly coupled under our experimental conditions. Repeating these experiments proved that our enzyme preparations consistently exhibited 92-96% coupling in the presence of L-arginine or 88-91% in the presence of NOHA. Oxygen consumption rates are calculated from the initial slope of oxygen traces in Fig. 5 and are listed in Table 2. Unlike the [O2]/[NADPH] ratio, enzyme preparations vary in terms of turnover numbers in a ±10% range.


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  		TABLE 1
Oxygen stoichiometry of eNOS The experiments were performed in the presence of excessive amounts of SOD and catalase. Theoretical values were derived from equations listed in the introduction, and the experimental values are averaged from multiple measurements of multiple eNOS holoenzyme preparations. Note that the [O2]/[NADPH] ratio is extremely close to the theoretical value in the absence of substrate, and eNOS is highly coupled in the presence of either L-arginine or NOHA.

 


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  		TABLE 2
Oxygenase activities of eNOS and nNOS The nNOS data are obtained from previous publications by Gao et al. (9), and the eNOS data were derived from the experiments shown in Fig. 5. At least four different enzyme preparations were tested for each isoform to give the range of activities.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The Uncoupled Reaction of eNOS—eNOS uncoupling is known to be controlled by substrate/cofactor availability (30, 32, 33), and the uncoupled reactions play important roles under various physiological/pathological conditions, such as atherosclerosis and septic shock (34-38). We applied the dHRP trapping technique with an NADPH regeneration system (9) to identify the initial product of the eNOS uncoupled reaction. Formula 10 is the direct product detected in the absence of substrate (L-arginine and/or NOHA). CaM accelerates eNOS-mediated Formula 10 generation by a factor of 3, which is consistent with the results reported by Masters and co-workers (30). In the absence of a trapping agent, H2O2 formed by spontaneous dismutation of Formula 10 will also be a detectable product.

In the absence of L-arginine, oxygen uptake by eNOS is very slow, ~4 min-1 in the presence of CaM, and even slower (~1 min-1) in its absence. NADPH-cytochrome P-450 reductase, a flavin enzyme that shares ~60% sequence identity with the eNOS reductase domain, has an uncoupled oxygen consumption rate of ~1 min-1 (measured in the present system).3 These results indicate that: 1) flavins in the NADPH-cytochrome P-450 reductase and eNOS are poor electron donors for molecular oxygen and 2) although slow, there are probably two sites for oxygen activation in eNOS, one in the reductase domain (presumably the flavins) (39) and the other in the oxygenase domain (presumably the heme), because binding of CaM to eNOS enables electron transfer from the reductase domain to oxygenase domain (1, 40) and activates overall oxygen consumption.

L-Arginine and BH4 Effects—The oxygen chemistry on the heme site of NOS has been generally assumed to follow that of cytochrome P-450 monooxygenases (1, 41-43). A simplified list of intermediates in the initial steps of heme-oxygen interaction can be represented as follows.


Formula 11REACTION 11
After these initial steps, activated oxygen can either heterolytically dissociate from heme to form superoxide anion,

Formula 12REACTION 12
or follow the cytochrome P-450 scheme to complete the monooxygenation of L-arginine.

Formula 13REACTION 13


Figure 4
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  		FIGURE 4.
BH4 effect. The reaction mixture contained 1800 µl of reaction buffer, 500 µM L-arginine, 440 µM NADPH, 23 µg/ml SOD, and 0.5 µM eNOS (BH4-free). A, BH4 was introduced after the reaction started. B, catalase was introduced after the reaction started. C, reaction was started in the presence of 5.5 µM BH2, and catalase was introduced after the reaction started. D, reaction was started in the presence of 5.5 µM BH4, and catalase was introduced after the reaction started.

 
The intermediates in Reaction 11 have been intensively studied with optical absorption spectroscopy, by stabilizing them under cryogenic conditions or by capturing them in real time with time-resolved measurements (19-21, 31, 44-47). With the heme known as the center for oxygen chemistry in NOS, we assume Reaction 12 is the major source for Formula 13 formation from the uncoupled eNOS in the presence of CaM (5, 9, 10, 30, 34, 39). However, the two intermediates (FeIIIO22- and FeIV=O) in Reaction 13 have not been convincingly observed to date, and it is unclear whether different NOS isoforms follow the same scheme in Reaction 13.

We report that the most striking difference between the oxygen metabolism of eNOS and nNOS is the L-arginine effect, wherein L-arginine strongly stimulates oxygen consumption of eNOS and inhibits that of nNOS (Fig. 3A). It is well known that recombinant nNOS is 5-7 times faster than eNOS in terms of NO production, and the difference is generally attributed to the rates at which the reductase domains provide electrons to the heme (25, 48). However, a comparison of the rates listed in Table 2 reveals a huge difference between eNOS and nNOS in oxygen consumption in the absence of L-arginine. So the question is: why is L-arginine-free eNOS so slow, and why is L-arginine-free nNOS so fast?

The slow oxygen consumption rate (~4 min-1) reported in the current study is the result of either inefficient FeIIO2 formation in the absence of L-arginine or a stabilized FeIIO2 complex that does not easily dissociate to form Formula 13 (Reactions 11 and 12). Tsai and co-workers (31) have studied the L-arginine effects on oxygen binding to eNOS in great detail, and their observations highly support the first hypothesis. Monitored at 427 nm, the accumulation of the FeIIO2 complex was shown to be much higher in the reaction with L-arginine than without (31), meaning that the presence of L-arginine facilitates the binding of molecular oxygen to ferrous heme in eNOS. More detailed kinetic data demonstrate a 7-8-fold slower association constant, as well as a 20-30-fold slower dissociation constant of oxygen in the presence of L-arginine, helping to explain the accumulation of FeIIO2 complex.


Figure 5
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  		FIGURE 5.
Stoichiometry of the eNOS reaction in the presence and absence of substrate. Standard reaction buffer and 7 µM CaM, 23 µg/ml SOD, 1.7 µM catalase, 5.5 µM BH4, and 1.5-5 µM eNOS were mixed before multiple injections of NADPH. A, no L-arginine or NOHA, 84.5 µM NADPH per step. B, 500 µM L-arginine as substrate, 84.5 µM NADPH per step. C, 500 µM NOHA as substrate after the carry-over L-arginine was consumed, 35.6 µM NADPH/step.

 
These kinetic data, although self-sufficient, are in agreement with the paradoxical L-arginine effects reported by other groups. On the one hand, the binding of L-arginine impedes the binding of diatomic molecules such as NO and CO as demonstrated by a variety of methods, including EPR (49-51), and it is logical to expect anti-cooperative binding of L-arginine and oxygen. On the other hand, L-arginine conversely stabilizes the FeIIO2 complex and slows down oxygen dissociation (52).

Up to now, there is no crystal structure for the O2-bound derivative of any of the NOS isoforms; thus, some details of oxygen binding geometry are still missing. Nevertheless, different groups have reported isoform-specific environments in the binding of diatomic molecules to nNOS and eNOS, using infrared/Raman spectroscopy and EPR (50, 53-57). All of these studies agree that despite the general similarity in structures, there are small differences among NOS isoforms that allow limited isoform specificity in the interaction between L-arginine and diatomic molecules, such as NO, CO, or O2.

A resonance Raman study by Rousseau and co-workers (58) presented evidence supportive of the differences in oxygen metabolism between eNOS and nNOS reported here. By monitoring the Fe-CO and C-O stretching modes of CO-ferrous complexes with bound L-arginine, the authors intriguingly showed that nNOS has a much more open substrate-binding pocket than eNOS. Regardless of the two possible interpretations proposed by this paper, the conclusion highly supports the tighter coupling of eNOS (Table 1) and the faster dissociation of superoxide from nNOS (Table 2).

Complementary electron-nuclear double resonance studies was performed by Hoffman, Masters, and co-workers (59-62), generating a series of publications regarding substrate binding to all three NOS isoforms. Based on spectral differences, the authors concluded that nNOS provides greater shielding of the charge on the guanidino nitrogen than eNOS does (63), which is one of the two interpretations in the previously mentioned resonance Raman study (58).

This differential L-arginine effect could be attributable to BH4. In the L-arginine-free eNOS, BH4 does not accelerate superoxide dissociation (Fig. 3A), but with nNOS, it was reported that BH4 accelerated the decay of the FeIIO2 complex by a factor of 70, irrespective of the presence of L-arginine (52).

Even though BH4 does not seem to directly affect oxygen binding in eNOS (31, 50), it controls the coupling of eNOS and, to some extent, the fate of oxygen reduction. The imperfect coupling of eNOS in the presence of substrate and absence of BH4 is evidenced not only in this study (Fig. 4), but also by other research groups using different approaches (33, 64, 65). In the presence of both substrate and BH4, however, eNOS is highly coupled (30, 64). The results presented herein (Fig. 4) confirm that BH4 is the "coupling switch" of eNOS; our approach is unique in that we quantitatively monitor oxygen consumption and utilize chemical reactions to convert accumulated ROS to visible and measurable molecular oxygen (Reactions 4, 6, 9, and 10).

BH2, a redox-incompetent BH4 analog, is unable to inhibit ROS formation by eNOS (Fig. 4C). Our results confirm the view that BH4 takes a redox role in the catalytic cycle, most likely by providing the second electron to the heme (21). Electron transfer from BH4 is proposed to be faster than that from the reductase domain and thus is consistent with the increased rate of oxygen uptake in the presence of BH4 over that in its absence (Fig. 4A). Under pathological conditions, the limited availability of BH4 has been shown to trigger eNOS uncoupling and increase the risk of cardiovascular diseases including atherosclerosis, coronary artery disease, and hypertension (66-73). In addition, external stimuli, such as the chemicals in cigarettes, can convert BH4 to BH2 in the human body (74-76). Our results support the view that the introduction or stabilization of BH4 may be used for the treatment of endothelial dysfunction (70-72, 77).

Stoichiometry—We report for the first time the quantitative measurement of eNOS oxygen stoichiometry. In the absence of L-arginine, the stoichiometry is simple, and the experimental values match the theoretical values (Table 1). Assessing the results from the simultaneous measurements (Fig. 2) and from the oxygen consumption experiment (Fig. 5A), it is clear that eNOS utilizes 1 mol of NADPH to provide electrons to 2 mol of molecular oxygen, generating 2 mol of superoxide anion as the product of the uncoupled reaction.

When L-arginine or NOHA is added in the presence of BH4, however, the eNOS reaction is highly coupled. A comparison with nNOS data (9) illustrates two points: 1) eNOS is more coupled than nNOS, with either L-arginine or NOHA as substrate and 2) in both eNOS and nNOS, the enzymes are more coupled with L-arginine as substrate than with NOHA. The first point can be easily understood, given the physiological importance of the precise control of blood pressure (41), and the second one is at least partially because of the substrate-specific H-bonding interactions with the terminal oxygen, as recently reported by Li et al. (53).

In conclusion, we have demonstrated the CaM, L-arginine, and BH4 effects on eNOS oxygen metabolism. We report, for the first time, the quantitative measurement of superoxide anion formation from eNOS. Our recombinant eNOS is 92-96% coupled in the presence of L-arginine, which strongly stimulates oxygen uptake. In the presence of either L-arginine or NOHA, the availability of BH4 controls eNOS coupling and diminishes ROS formation by eNOS. This exquisite control is necessitated by its role in the vascular system in which the production of ROS must be prevented. It is verified that oxygen consumption by eNOS in the absence of substrate is extremely low, and only when substrate is supplied does NO production proceed in a highly coupled, physiologically significant process.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants GM52419 and HL30050 (to B. S. S. M.), Grant AQ-0012 for the Robert A. Welch Foundation Distinguished Chair in Chemistry (to B. S. S. M.), Grant Agency of Czech Republic (GACR) Grant 303/05/0336, and Ministry of Education, Youth and Sports (MSMT) Grant 1M0520 (to P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229. Tel.: 210-567-6627; E-mail: masters{at}uthscsa.edu.

2 The abbreviations used are: NOS, nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; BH4, tetrahydrobiopterin; BH2, 7,8-dihydrobiopterin; dHRP, diacetyldeuteroheme-substituted horseradish peroxidase; NADPH, nicotinamide adenine dinucleotide phosphate; ROS, reactive (or reduced) oxygen species; NOHA, N{omega}-hydroxy-L-arginine; IDP-2, cytosolic NADP+-dependent isocitrate dehydrogenase; CaM, Ca2+/calmodulin; SOD, superoxide dismutase. Back

3 Y. T. Gao, L. J. Roman, P. Martásek, S. P. Panda, Y. Ishimura, and B. S. S. Masters, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Christopher C. Marohnic (Postdoctoral Fellow in Dr. Masters' laboratory, The University of Texas Health Science Center at San Antonio) for helpful discussions and Karen McCammon for preparing IDP-2.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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