Mechanism and Kinetics of Inducible Nitric Oxide Synthase Auto-S-Nitrosation and Inactivation

Abstract

Nitric oxide (NO), the product of the nitric oxide synthase (NOS) reaction, was previously shown to result in S-nitrosation of the NOS Zn²⁺-tetrathiolate and inactivation of the enzyme. To probe the potential physiological significance of NOS S-nitrosation, the inactivation timescale of the inducible NOS isoform (iNOS) was determined and found to directly correlate with an increase in iNOS S-nitrosation. A kinetic model of NOS inactivation in which arginine is treated as a suicide substrate was developed. In this model, NO synthesized at the heme cofactor is partitioned between release into solution (NO release pathway) and NOS S-nitrosation followed by NOS inactivation (inactivation pathway). Experimentally determined progress curves of NO formation were fit to the model. The NO release pathway was perturbed through addition of the NO traps oxymyoglobin (MbO₂) and β2 H-NOX, which yielded partition ratios between NO release and inactivation of ~100 at 4 μM MbO₂ and ~22,000 at saturating trap concentrations. The results suggest that a portion of the NO synthesized at the heme cofactor reacts with the Zn²⁺-tetrathiolate without being released into solution. Perturbation of the inactivation pathway through addition of the reducing agents GSH or TCEP resulted in a concentration-dependent decrease in iNOS S-nitrosation that directly correlated with protection from iNOS inactivation. iNOS inactivation was most responsive to physiological concentrations of GSH with an apparent K_m value of 13 mM. NOS turnover that leads to NOS S-nitrosation might be a mechanism to control NOS activity, and NOS S-nitrosation could play a role in the physiological generation of nitrosothiols.

Nitric oxide (NO) plays essential and disparate roles in mammalian physiology, causing vasodilation in the cardiovascular system, neurotransmission in nervous tissue, and cytotoxicity against pathogens in the immune response. NO is synthesized by nitric oxide synthase (NOS), which catalyzes the conversion of arginine to citrulline and NO, using oxygen (O₂) and NADPH as cosubstrates (1). Mammals possess three NOS isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). As a signaling agent, low nanomolar NO concentrations are synthesized by eNOS or nNOS. As a cytotoxin, micromolar NO concentrations are generated by iNOS at sites of infection or inflammation (2).

All three NOS isoforms are dimeric and share a common architecture, composed of two domains: a reductase and an oxidase domain. The reductase domain contains the flavin cofactors FAD and FMN and a binding site for co-substrate NADPH, which shuttle electrons from NADPH to the oxidase domain of the opposite monomer in the dimer during catalysis. Therefore, NOS is only active as a homodimer. The oxidase domain binds the substrate arginine as well as the heme and tetrahydrobiopterin (H₄B) cofactors. The interface of the oxidase domain homodimer contains a Zn²⁺-tetrathiolate that is comprised of two cysteines from each monomer.

In its classical signaling role, NO is captured by the heme cofactor of soluble guanylate cyclase (sGC), activating sGC to produce the secondary messenger cyclic GMP (cGMP) (3). However, mounting evidence points toward an alternative, cGMP-independent NO signaling pathway where the S-nitrosation of cysteine residues regulates protein structure and activity (4). S-nitrosation has been implicated in a broad spectrum of diseases including cancer, diabetes, and other cardiovascular, pulmonary, and neurological disorders (4). Yet the mechanism by which nitrosothiols are formed in vivo is unknown.

The direct transfer of a nitrosyl group from a nitrosothiol to a free thiol (i.e. transnitrosation) is a simple bimolecular reaction. However, the initial formation of a nitrosothiol from NO and a free thiol requires a one-electron oxidation. Transnitrosation reactions therefore necessitate an “initiating” nitrosothiol formed by a protein that uses NO and a free thiol to synthesize a nitrosothiol with the aid of an oxidant. The mitochondrial hemoprotein cytochrome c was recently shown to satisfy these criteria using NO, glutathione (GSH), and ferric heme to form S-nitrosoglutathione (GSNO) (5, 6). However, the high K_m value of NO and the high protein concentration utilized to observe this reaction suggests that another protein is responsible for the physiological generation of nitrosothiols at the lower NO concentrations commonly found in vivo. Cytochrome c may play a more significant role under the high NO levels encountered during nitrosative stress.

NOS is a potential candidate for the initial formation of nitrosothiols as all three mammalian NOS isoforms selectively form nitrosothiols at their Zn²⁺-tetrathiolate cysteines (7–11). iNOS S-nitrosation dissociates the iNOS dimer, inactivates the enzyme, and may expose the nitrosothiols for transnitrosation reactions (8). If involved in S-nitrosation, NOS must balance participation in this pathway against enzyme inactivation. Of the three NOS isoforms, only iNOS has been shown to participate in protein–protein interaction mediated S-nitrosation reactions. Kim et al. showed that formation of an iNOS-COX-2 complex was required for S-nitrosation of cyclooxygenase-2 (COX-2) (12). Furthermore, procaspase-3 and iNOS participate in an NO-dependent protein-protein interaction (13). As caspase-3 is known to be nitrosated on its active-site cysteine (14), iNOS might directly transnitrosate caspase-3. Additionally, a protein–protein complex between iNOS and arginase 1 was shown to be necessary for arginase 1 S-nitrosation (15).

Once formed, nitrosothiols can be transferred to downstream targets by formation of specific protein-protein or protein-small molecule interactions in a process analogous to phosphoryl transfer (16). Mirroring the role of ATP in phosphorylation, transnitrosation reactions may involve a small molecule nitrosothiol donor. GSH is the most abundant thiol in mammalian cells (physiological concentrations range from 0.5 to 10 mM) (17), and GSNO has been detected both intra- and extracellularly (18–20). Furthermore, GSNO can specifically transnitrosate thioredoxin (21, 22) and GSNO reductase knockout mice have markedly increased levels of S-nitrosated proteins (4). As NOS is S-nitrosated and can participate in transnitrosation reactions, NOS may also be responsible, at least in part, for the physiological generation of GSNO.

Here, we performed a detailed kinetic analysis of iNOS S-nitrosation and inactivation by treating arginine as a suicide substrate. This model allowed direct determination of the partition ratio between NO release into solution and iNOS S-nitrosation followed by inactivation. Further insight was gained by perturbing this partition ratio through varying the concentration of arginine, NO traps, or reductants. Our results indicate that the main mechanism of iNOS inactivation is S-nitrosation of the Zn²⁺-tetrathiolate. We hypothesize that a tunnel, present in all three NOS isoforms, acts as a conduit between the heme and Zn²⁺-tetrathiolate and facilitates this S-nitrosation. Our kinetic results have implications on iNOS S-nitrosation as an initial source of nitrosothiols such as GSNO.

Experimental Procedures

General materials

All chemicals used were of the highest purity commercially available and were purchased from Sigma (St. Louis, MO, USA), Aldrich (Milwaukee, WI, USA), or Fisher Scientific (Pittsburgh, PA, USA) unless otherwise noted below.

Protein expression, purification, and preparation

Expression and purification of murine iNOS (coexpressed with calmodulin) (8, 23) and β2 H-NOX (24) were carried out as described previously. iNOS concentrations were determined using the method of Bradford with bovine serum albumin as the standard (25). Horse heart myoglobin was purchased from Sigma (St. Louis, MO). Horse heart oxymyoglobin (MbO₂) was generated through dithionite reduction and desalting as described previously for oxyhemoglobin (26). Oxymyoglobin concentrations were determined using a heme extinction coefficient of 13.9 mM⁻¹cm⁻¹ at 542 nm (27). Generation of horse heart apomyoglobin by acidification to pH 2 with 0.1 N HCl and heme extraction with methyl ethyl ketone was performed as described previously (28). Apomyoglobin concentrations were determined using an extinction coefficient of 15.5 mM⁻¹cm⁻¹ at 280 nm (29).

iNOS activity assays

Horse heart MbO₂ assays were carried out as previously described for NOS oxyhemoglobin assays (26) with the following modifications. This assay monitors the absorbance change upon reaction of MbO₂ with NO to form nitrate and metmyoglobin (metMb). Assays were monitored at either 401, 540, or 581 nm in 300 μL total volumes in clear 96-well microplates. Due to the strong absorbance of myoglobin at 401 nm, assays with MbO₂ concentrations greater than 15 μM were monitored at 540 or 581 nm. Activities were determined using extinction coefficients of 45.5 mM⁻¹cm⁻¹ for the increase in absorbance at 401 nm, 7.5 mM⁻¹cm⁻¹ for the decrease in absorbance at 540 nm, or 10.5 mM⁻¹cm⁻¹ for the increase in absorbance at 581 nm. These extinction coefficients were determined by the difference spectrum of horse heart MbO₂ and metMb (formed by reaction of MbO₂ with NO) at known initial MbO₂ concentrations.

Biotin switch method

The biotin switch method (11) was employed to assay S-nitrosation of iNOS. To avoid nitrosothiol decomposition, samples were protected from direct sunlight as much as possible. Assays contained 1–4 μM iNOS, 5 mM Arg, 2 mM NADPH, 50 mM NaCl, with or without GSH/TCEP in either 50 mM HEPES or HEN buffer (250 mM HEPES, 2 mM EDTA, 0.2 mM neocuproine) at pH 7.5 in 100 μL total volumes. iNOS S-nitrosation was initiated by NADPH addition and quenched by addition of 100 μL blocking buffer (10% SDS and 60 mM N-ethylmaleimide in HEN buffer) and incubated at 55 °C for 30 minutes. Protein was then precipitated with 1.2 mL acetone pre-cooled to −20 °C and pelleted at 14,000 rpm for 10 minutes at 4 °C. The supernatant was removed and the pellet was washed with 0.9 mL more acetone, and the pellets were air-dried. Dried pellets were resuspended in PBS with 5% SDS, 30 mM ascorbate, and 500 μM biotin-maleimide or biotin-iodoacetamide (Thermo Fisher Scientific; Rockford, IL) and incubated at 37 °C for 30 minutes. Biotin labeling was quenched with 50 mM DTT or gel loading buffer containing 10 mM DTT. Relative iNOS S-nitrosation levels were determined by immunoblotting for biotin labeling using the Vectastain ABC Kit (Vector laboratories; Burlingame, CA) and SuperSignal West pico or femto maximum sensitivity substrate (Thermo Fisher Scientific; Rockford, IL). To ensure equal protein loading between samples, the blots were stained with Ponceau stain after imaging.

Results

Nitrosation and inactivation of iNOS during enzymatic turnover

To gain insight into the physiological role of NOS S-nitrosation, we first determined if there was a direct temporal correlation between iNOS S-nitrosation and inactivation. While iNOS was previously shown to be S-nitrosated by exogenously added NO (8) and endogenously generated NO at the heme cofactor (30), the timescale of auto-S-nitrosation were not investigated. Here, iNOS was rapidly S-nitrosated by NO generated during the catalytic reaction (Figure 1A), and the level of iNOS auto-S-nitrosation peaked at ~30 minutes and did not increase at 60 minutes. S-nitrosation levels were determined using the biotin switch assay (11) which selectively and covalently labels sites of S-nitrosation with biotin for detection by immunoblotting.

Figure 1.

iNOS S-nitrosation and inactivation during enzymatic turnover. (A) iNOS S-nitrosation during enzymatic turnover. Each reaction contained 4 μM iNOS, 5 mM Arg, 2 mM NADPH, 50 mM NaCl, and 50 mM HEPES pH 7.5. iNOS activity was initiated by NADPH addition and 100 μL aliquots were taken at the indicated timepoints and quenched with an equal volume of 250 mM HEPES at pH 7.4 containing 2 mM EDTA, 0.2 mM neocuproine, 60 mM N-ethylmaleimide, and 10% SDS. Relative levels of iNOS S-nitrosation were determined by the biotin switch method (see Experimental Procedures). (B) iNOS inactivation during enzymatic turnover. Two separate reactions were initiated containing 90 nM iNOS, 15 mM Arg, 150 mM NaCl, and 100 mM HEPES pH 7.5. To one reaction iNOS activity was initiated at time zero by NADPH (600 μM) addition (black bars), to the other reaction NADPH was only added immediately prior to rate determination (grey bars). At the given timepoints, 100 μL aliquots of each reaction were added to 200 μL of 45 μM MbO₂ in 100 mM HEPES pH 7.5 to obtain final concentrations of 30 nM iNOS, 5 mM Arg, 50 mM NaCl, 30 μM MbO₂, and 100 mM HEPES pH 7.5.

Exogenous addition of NO was previously shown to inactivate NOS in vitro (8, 31–33). This inactivation correlated with iNOS dimer dissociation due to S-nitrosation of the Zn²⁺-tetrathiolate (8). However, the timescale of inactivation by endogenously generated NO at the heme cofactor had not been investigated. To determine the timescale of iNOS auto-inactivation, iNOS-catalyzed NO formation (and thus auto-S-nitrosation) was initiated by addition of both Arg and NADPH at time zero and the remaining iNOS activity at 5, 15, and 30 minutes was determined using the increase in absorbance at 401 nm upon reaction of NO with oxymyoglobin (MbO₂) to form nitrate and metmyoglobin (metMb). Indeed, iNOS catalyzed NO formation resulted in rapid loss of NOS activity (Figure 1B black bars) that correlated with an increase in iNOS auto-S-nitrosation (Figure 1A). A similar rapid decline in nNOS activity was previously observed with near complete inactivation after 30–90 minutes (34, 35). To ensure the observed iNOS inactivation was a direct result of NOS activity and not general protein instability at room temperature, only Arg was added at time zero and NADPH was not added until immediately prior to assaying the rate at each timepoint. A relatively modest loss in iNOS activity was observed under these conditions (Figure 1B grey bars) indicating that iNOS auto-inactivation was a direct result of NOS activity and auto-S-nitrosation.

Importantly, addition of superoxide dismutase (SOD) (50 units) and catalase (50 units) failed to protect iNOS from inactivation and instead resulted in a slight (~15%) increase in iNOS inactivation (data not shown). A similar slight increase in inactivation rate was previously observed upon addition of SOD to iNOS (36) and eNOS (31) activity assays. This result indicated that iNOS auto-inactivation was not due to generation of reactive oxygen species from uncoupling of the iNOS reductase and oxidase domains.

Kinetic model of iNOS auto-inactivation

The observation that iNOS auto-inactivation directly correlated with an increase in iNOS auto-S-nitrosation led us to develop a kinetic model for iNOS inactivation by S-nitrosation (Figure 2A). In this model Arg binding to iNOS (k₁ and k₂) is followed by irreversible formation of NO and citrulline (k₃) (NO formation pathway). The NO formed at the heme cofactor is then released into solution (k₅ and k₆) where NO then reacts with MbO₂ to form nitrate and metMb (k₇) (NO release/detection pathway). Alternatively, NO may react with the Zn²⁺-tetrathiolate resulting in S-nitrosation (k₉) followed by dimer dissociation and inactivation (k₁₁) (S-nitrosation/inactivation pathway). Prior to dimer dissociation, S-nitrosation can be reversed (k₁₀′) through addition of exogenous reductants (see below). Note that E•NO represents NO sequestered within iNOS such that the NO is inaccessible to MbO₂, but not necessarily bound to the heme iron. Therefore, k₆ represents the diffusion of NO into the iNOS protein environment, not NO binding to the heme iron. The kinetic model in Figure 2A can be further simplified by replacing the NO release/detection and inactivation pathways with net rate constants (Figure 2B):

$${k_{5,7}}^{\prime }=\frac{k_5{k}_7[{\mathit{MbO}}_2]}{k_5+k_6+k_7[{\mathit{MbO}}_2]}$$ (1)

$${k_{9,11}}^{\prime }=\frac{k_9{k}_{11}}{k_9+{k_{10}}^{\prime }+k_{11}}$$ (2)

Figure 2.

Kinetic model of iNOS inactivation. (A) Complete kinetic model of iNOS auto-inactivation by S-nitrosation and dimer dissociation where the kinetic readout is reaction of NO with MbO₂. Gray boxes indicate the NO formation, inactivation, and NO release/detection pathways. E represents the enzyme (i.e. iNOS), R represents arginine, E•R represents arginine bound within the iNOS active-site, E•NO represents nitric oxide sequestered within iNOS but not necessarily bound to the heme iron, E-SNO represents iNOS S-nitrosated at the Zn²⁺-tetrathiolate cysteines, and E_i represents inactivated iNOS. (B) A simplified kinetic model in which the inactivation and NO release/detection pathways are represented by net rate constants. (C) General kinetic model of a suicide substrate where S represents the substrate and P represents the product.

The kinetic model in Figure 2B is identical to that of a suicide substrate (mechanism-based inhibitor) (Figure 2C). Therefore, using suicide substrate analysis (37–43), plots of NO formation over time may be fit to equation 3:

$$[NO]={[NO]}_{\infty }(1-e^{-\lambda t})$$ (3)

where [NO]_∞ is the total concentration of NO formed at infinite time and λ is the NOS inactivation rate. Equation 3 provided excellent fits to progress curves of iNOS catalyzed NO formation obtained using the MbO₂ assay (Figure 3). MbO₂ rapidly reacts with NO to form nitrate and metMb at a rate of 3.4 × 10⁷ M⁻¹s⁻¹ (44), which may be followed spectrally by a change in absorbance at 401, 540, or 581 nm. The timescale of inactivation in the MbO₂ assay (Figure 3) was slower than without MbO₂ (Figure 1B), since MbO₂ reacts with NO to form nitrate and metmyoglobin (metMb) further indicating that NO is the primary source of iNOS inactivation.

Figure 3.

Progress curve of NO formation exhibits inactivation over time. The reaction contained 15 nM iNOS, 5 mM Arg, 200 μM NADPH, 50 mM NaCl, 4 μM MbO₂, and 100 mM HEPES pH 7.5 in 300 μL total volume in a 96-well microplate. NO formation was determined by the increase in absorbance at 401 nm observed upon reaction of NO with MbO₂ to form metMb and nitrite. Data was fitted to equation 3 and the resulting fit is shown as a solid black line. The dashed line represents the linear fit to the first five minutes of the progress curve.

Using suicide substrate analysis (37–43) [NO]_∞ and λ can be expressed in terms of the kinetic model rate constants:

$$\lambda =\frac{k_3{k_{9,11}}^{\prime }}{k_3+{k_{5,7}}^{\prime }+{k_{9,11}}^{\prime }}$$ (4)

$${[NO]}_{\infty }=r[\mathit{iNOS}]=\frac{{k_{5,7}}^{\prime }}{{k_{9,11}}^{\prime }}[\mathit{iNOS}]$$ (5)

where r is the partition ratio between the NO release/detection and S-nitrosation/inactivation pathways. This partition ratio can easily be determined for a given set of conditions via a plot of [NO]_∞ versus the initial iNOS concentration. If Arg is a suicide substrate, then this plot is predicted to give a straight line that intersects the origin with a slope equal to r. Indeed, fitting a plot of [NO]_∞ versus iNOS concentration at saturating substrate concentrations and 4 μM MbO₂ yielded a straight line that passed through the origin with a slope of 106 ± 2 (Figure S1). This indicated that ~100 molecules of NO were released into solution and reacted with MbO₂ for every one molecule of NO that led to S-nitrosation of the Zn²⁺-tetrathiolate. However, it should be noted that the [NO]_∞ value and therefore the r-value greatly depend on the MbO₂ concentration utilized in the assay. This ability of MbO₂ to “trap” solution NO and alter the inactivation rate will be exploited below.

Reductants protect iNOS from S-nitrosation and inactivation

Thiols like glutathione (GSH) and other reducing agents such as TCEP are capable of reducing nitrosothiols to free thiols. GSH and other thiol reducing agents were shown to prevent iNOS (45, 46) and nNOS inactivation (35, 47). Therefore, the inactivation pathway of the kinetic model (Figure 2A) can be perturbed through addition of reducing agents. Indeed, both GSH (Figure 4A) and TCEP (Figure 4B) reduced the inactivation rate, though TCEP required approximately two orders of magnitude lower concentration compared to GSH for a similar effect.

Figure 4.

Protection of iNOS inactivation by reductants. Progress curves of iNOS catalyzed NO formation in the presence of increasing concentrations of (A) GSH and (B) TCEP. Individual reactions contained 15 nM iNOS, 5 mM Arg, 200 μM NADPH, 50 mM NaCl, 4 μM MbO₂, 100 mM HEPES pH 7.5, and varying concentrations of either GSH or TCEP in 300 μL total volumes in a 96-well microplate. NO formation was measured via the change in absorbance at 401 nm upon reaction of MbO₂ with NO. (C) Plot of [NO]_∞ versus GSH (circles, solid line) or TCEP (squares, dashed line) concentration. Data was fitted using equations 2, 5, and 6. (D) Plot of inactivation rate (λ) versus GSH (circles, solid line) or TCEP (squares, dashed line) concentration. Data was fitted using equations 2, 4, and 6.

As only a finite concentration of S-nitrosated iNOS (E-SNO in Figure 2A) exists, we hypothesized that reducing agent addition would exhibit saturation kinetics and alter the rate of nitrosothiol reduction (k₁₀′) through the following relationship:

$${k_{10}}^{\prime }=\frac{k_{10}[\mathit{reductant}]}{K_{m,\mathit{app}}^{\mathit{red}}+[\mathit{reductant}]}$$ (6)

where k₁₀ is the maximum rate of nitrosothiol reduction, [reductant] is the GSH or TCEP concentration, and $K_{m,\mathit{app}}^{\mathit{red}}$ is the apparent K_m of nitrosothiol reduction. Therefore, when no reductant is added to the assay mixture, k₁₀′ = 0 and nitrosothiol formation is irreversible and committed to enzyme inactivation. Plots of [NO]_∞ versus [reductant] yielded excellent fits to the relationship between [NO]_∞ and [reductant] shown in equation 7 derived from equations 2, 5, and 6 (Figure 4C).

$${[NO]}_{\infty }={k_{5.7}}^{\prime }[\mathit{iNOS}]\left(\frac{1}{k_9}+\frac{1}{k_{11}}\right)+\left(\frac{{k_{5,7}}^{\prime }{k}_{10}[\mathit{iNOS}]}{k_9{k}_{11}}\right)\left(\frac{[\mathit{reductant}]}{K_{m,\mathit{app}}^{\mathit{red}}+[\mathit{reductant}]}\right)$$ (7)

Similarly, plots of λ versus [reductant] concentration also yielded excellent fits to the relationship derived from equations 2, 4, and 6 (Figure 4D). These experiments and those below allowed estimation of values for the various kinetic constants in the kinetic model. In general, data plots were fitted using non-linear regression in Kaleidagraph (Synergy Software; Reading, PA) and the resulting kinetic constants are displayed in Table 1.

Table 1.

Rate constants derived from the kinetic model of inactivation.

Rate constant	Kinetic Model Step	Reductant titration	Varying MbO2
k3 (s−1)	NO formation	NA	~2.2a
k5 (s−1)	NO diffusion from NOS	NA	16.1 ± 5.6b
k6[NO] (s−1)	NO diffusion into NOS	NA	(1.6 ± 0.6) × 103 b
k7 (M−1s−1)	MbO2 reaction with NO	3.4 × 107 c	3.4 × 107 c
$\frac{{\text{k}}_9{\text{k}}_{11}}{{\text{k}}_9+{\text{k}}_{11}}({\text{s}}^{-1})$	Inactivation pathway without reductant	(6.7 ± 0.2) ± 10−4 d	(7.4 ± 2.5) × 10−4 b
$\frac{{\text{k}}_9{\text{k}}_{11}}{{\text{k}}_9+{\text{k}}_{10}+{\text{k}}_{11}}({\text{s}}^{-1})$	Inactivation pathway with saturating reductant	(1.1 ± 0.1) ± 10−4 d	NA
k10GSH/k10TCEP	S-nitrosothiol reduction	2.0 ± 0.2d	NA
Km, appGSH (μM)		(1.3 ± 0.2) × 104 d	NA
Km, appTCEP (μM)		(1.4 ± 0.1) × 102 d	NA

^aRate was approximated from the single turnover rate of NHA formation from arginine, the single turnover rate of NO, citrulline formation from NHA, and the rate of NO release from iNOS ferric heme (50–54).

^bRate was determined from fit in Figure 7A.

^cRate from reference (44).

^dRate was determined from fit in Figure 5C.

If iNOS auto-S-nitrosation is the main source of auto-inactivation, protection from inactivation by reductants should correlate with a decrease in S-nitrosation. Indeed, using the biotin switch assay, a decrease in iNOS S-nitrosation was observed with increasing concentrations of both GSH and TCEP (Figure 5). In particular, a concentration-dependent decrease in iNOS auto-S-nitrosation was observed at GSH concentrations greater than 1 mM and TCEP concentrations greater than 30 μM. Importantly, the effect of both reductants on iNOS S-nitrosation correlated well with the observed decrease in inactivation rate under varying reductant concentrations (Figure 4D).

Figure 5.

Protection of iNOS auto-S-nitrosation by reductants. (A) The anti-biotin blot shows a dose-dependent decrease in iNOS S-nitrosation upon addition of GSH and TCEP. (B) Quantitation of anti-biotin blots with increasing concentrations of GSH (black bars) and TCEP (grey bars). Levels of iNOS S-nitrosation were normalized to the level observed with no added reductant. Error bars represent standard deviation from the mean. Each reaction contained 2 μM iNOS, 5 mM Arg, 2 mM NADPH, 50 mM NaCl, and varying concentrations of either GSH or TCEP in HEN buffer (250 mM HEPES, 2 mM EDTA, 0.2 mM neocuproine, pH 7.5) in 100 μL total volume. iNOS activity was initiated by NADPH addition and quenched after 20 min in HEN buffer with 60 mM N-ethylmaleimide and 10% SDS. Relative levels of iNOS S-nitrosation were determined by the biotin switch method (see Experimental Procedures).

The kinetic model described above (Figure 2) indicates iNOS is only inactivated upon dimer dissociation (k₁₁) following S-nitrosation (k₉). If this model is correct, then added reductants (k₁₀′) should protect S-nitrosated iNOS (E-SNO) from inactivation, but not recover activity from inactivated iNOS (E_i) as k₁₁ is irreversible. To determine if the activity of inactivated iNOS could be recovered by reductant addition, iNOS was fully inactivated as in Figure 1B at which point 3 mM GSH or 1 mM TCEP was added and the resulting iNOS activity was assayed. iNOS activity was not recovered by this method (data not shown) indicating that iNOS inactivation is irreversible. This result was consistent with a previous study in which GSH was added to fully inactivated nNOS and no recovery of nNOS activity was observed (35). Therefore, k₁₁ is a necessary irreversible rate constant as part of the kinetic model (Figure 2A). The inability to recover inactivated iNOS is also consistent with the hypothesis that dimer dissociation following S-nitrosation and Zn²⁺ loss, not S-nitrosation or Zn²⁺ loss itself, is responsible for inactivation.

iNOS inactivation rate depends on the rate of NO formation

Further insight into the kinetic model was obtained through perturbation of the NO formation pathway (Figure 2A). This was done by utilization of subsaturating Arg concentrations. When Arg is not saturating [NO]_∞ is governed by equation 8 (37):

$${[NO]}_{\infty }=\frac{{k_{5,7}}^{\prime }[\mathit{iNOS}][\mathit{Arg}]}{{k_{9,11}}^{\prime }\left([\mathit{Arg}]+\frac{k_3+{k_{5,7}}^{\prime }+{k_{9,11}}^{\prime }}{k_3}{K}_m\right)}=\frac{{k_{5,7}}^{\prime }[\mathit{iNOS}][\mathit{Arg}]}{{k_{9,11}}^{\prime }([\mathit{Arg}]+K_{m,\mathit{app}})}$$ (8)

where K_m is the Michaelis constant for Arg previously determined to be 8.6 μM (48). Note that when Arg is saturating equation 9 reduces to equation 5. Also, note that the apparent K_m value for Arg (K_m,app) is larger than the actual K_m value by a factor of (k₃ + k_5,7′ + k_9,11′)/k₃. Progress curves at varying Arg concentrations from 6.25 to 100 μM were fitted to equation 3 to determine [NO]_∞ and then plots of [NO]_∞ versus Arg concentration (Figure S3) were fitted to equation 9 by non-linear regression using Kaleidagraph (Synergy Software; Reading, PA) to obtain the k_5,7′/k_9,11′ ratio and apparent K_m value. Using 8 μM MbO₂, an apparent K_m value of 42 ± 2 μM and k_5,7′/k_9,11′ ratio of 690 ± 20 was determined. Alternatively, equation 8 may be rearranged to give equation 9:

$$\frac{[\mathit{iNOS}]}{{[NO]}_{\infty }}=\frac{{k_{9,13}}^{\prime }}{{k_{5,7}}^{\prime }}+\frac{{k_{9,11}}^{\prime }{K}_{m,\mathit{app}}}{{k_{5,7}}^{\prime }[\mathit{Arg}]}$$ (9)

Therefore, the plot of [iNOS]/[NO]_∞ versus 1/[Arg] was predicted to yield a straight line with an ordinate intercept of k_9,11′/k_5,7′ and a slope of k_9,11′K_m/k_5,7′. Indeed, the plot of [iNOS]/[NO]_∞ versus 1/[Arg] yielded a straight line (Figure S3 inset) with a k_9,11′/k_5,7′ value of 0.0011 ± 0.0002 and a k_9,11′K_m/k_5,7′ value of 0.069 ± 0.002. The positive slope observed highlighted the difference between Arg and a classical suicide substrate as the plot of [E]/[P]_∞ versus 1/[S] for a classical suicide substrate would yield horizontal line since [P]_∞ is independent of substrate concentration. A classical suicide substrate binds the enzyme and then reacts within the active site in a manner that prevents subsequent substrate binding and turnover. In contrast, NO inactivates iNOS at a site distal to the active site (i.e. the Zn²⁺-tetrathiolate). Thus, Arg binding and turnover proceeds until iNOS S-nitrosation, dimer dissociation, and enzyme inactivation occurs and [NO]_∞ depends on the relative flux between the NO formation and inactivation pathways at subsaturating Arg concentrations.

NO traps decrease the iNOS inactivation rate

Finally, varying concentrations of solution NO traps were used to perturb the NO release/detection pathway (Figure 2A). Previously, oxyhemoglobin (HbO₂) was utilized as a solution NO trap to protect nNOS (32, 33) and eNOS (31, 49) from auto-inactivation in activity assays. Here, in place of hemoglobin, horse heart myoglobin was utilized since it lacks cysteine residues and thus avoids potential assay artifacts due to reaction of NO or nitrosothiols with cysteines. Using saturating Arg, the inactivation rate was determined at varying concentrations of MbO₂ from 4–160 μM. The resulting plot of λ versus MbO₂ concentration exhibited a dependence on the amount of trap at low MbO₂ concentrations but was independent at high MbO₂. The trap-dependent portion of the plot is likely due to competition between MbO₂ and the Zn²⁺-tetrathiolate for reaction with NO in solution; the trap-independent portion likely indicates that a portion of NO reacts with the Zn²⁺-tetrathiolate without first being released into solution (see Discussion). This concentration-dependent trap result was predicted from the kinetic model, and plots of λ versus MbO₂ concentration yielded excellent fits to the relationship between λ and MbO₂ derived from equations 1 and 4 (Figure 6A). To simplify the determination of individual kinetic constants in the kinetic model, the plot of 1/λ versus MbO₂ concentration (Figure 6A inset) was fitted to equation 10 using Kaleidagraph (Synergy Software; Reading, PA).

Figure 6.

Plots of iNOS inactivation rate (λ) versus (A) MbO₂ concentration and (B) β2 H-NOX concentration. Individual reactions contained 15 nM iNOS, 5 mM Arg, 200 μM NADPH, 50 mM NaCl, 100 mM HEPES pH 7.5, and varying concentrations of either MbO₂ or β2 H-NOX in 300 μL total volumes in a 96-well microplate. NO formation was measured as described under Experimental Procedures. The resulting progress curves were fitted to equation 3 to determine the inactivation rate (λ). The plots of inactivation rate (λ) versus NO trap concentration were fitted using equations 1 and 4. The inset in (A) was fitted to equation 8.

$$\frac{1}{\lambda }=\left(\frac{1}{k_3}+\frac{1}{{k_{9.11}}^{\prime }}\right)+\left(\frac{k_5}{k_3{k_{9.11}}^{\prime }}\right)\left(\frac{k_7[{\mathit{MbO}}_2]}{k_5+k_6+k_7[{\mathit{MbO}}_2]}\right)$$ (10)

Since k₃ could be estimated from published single-turnover rates of heme reduction (50), Arg oxidation (51), and NO formation from N-hydroxyarginine (52–54), the plot of 1/λ versus [MbO₂] allowed direct determination of k₅, k₆, and k_9,11′ (Table 1).

To ensure that the effect of MbO₂ on the inactivation rate was not due to some property of MbO₂ other than the ability to trap solution NO, the ability of apomyoglobin and metmyoglobin (metMb) to protect iNOS from inactivation was tested. The addition of increasing concentrations of apomyoglobin or metMb to 5 μM MbO₂¹ both failed to decrease the inactivation rate (data not shown). Similarly, addition of metHb had no effect on nNOS (33) or eNOS (31) inactivation in previous studies. In addition, inhibition of iNOS by the nitrate generated from the reaction of NO with MbO₂ could not account for the decrease in inactivation rate, as nitrate concentrations up to 1 mM did not alter iNOS activity (data not shown). The lack of iNOS inhibition by nitrate was in line with previous reports examining nitrite and nitrate inhibition of nNOS (33, 34). Taken together, these observations indicated that the ability of increasing MbO₂ concentrations to decrease the inactivation rate was solely due to an increased ability to trap solution NO.

To assess whether the trap-independent inactivation rate observed at high MbO₂ concentrations was due to a thiol-dependent process (i.e. S-nitrosation) or due to other potential, independent inactivation processes, the concentration of NO traps were varied in the presence of 10 mM GSH. If trap-dependent and trap-independent inactivation were both a result of iNOS S-nitrosation, then addition of GSH would result in a horizontal line lying below the trap-independent inactivation rate in the plot of λ versus MbO₂ concentration. However, 10 mM GSH interfered with the MbO₂ assay at high MbO₂ concentrations². Therefore, an alternative method to trap and detect NO without generating a ferric heme species was sought. The heme-domain (residues 1–217) of the β2 subunit of soluble guanylate cyclase (β2 H-NOX) is ideal in this respect as it is a native NO receptor, does not bind oxygen, is stable to oxidation, and binds NO with a characteristic spectral shift (Soret λ_max 433 nm to 399 nm) (24). Therefore, the β2 H-NOX concentration was varied in a similar fashion to MbO₂ and inactivation rates were determined in the presence and absence of 10 mM GSH. Plots of λ versus β2 H-NOX concentration were similar to those using MbO₂ (Figure 6B) with one important distinction: the concentration of β2 H-NOX necessary to reach the trap-independence was lower than with MbO₂³. This correlates with the more rapid binding of NO to β2 H-NOX (>1.4 × 10⁸ M⁻¹s⁻¹) compared to the reaction of NO with MbO₂ (3.4 × 10⁷ M⁻¹s⁻¹) (44, 55) and further indicates that the observed decreases in inactivation rate were due to trapping solution NO. As hypothesized above, addition of 10 mM GSH resulted in constant inactivation rates over the entire range of β2 H-NOX concentrations, with an inactivation rate below the trap-independent inactivation rate (Figure 6B). This indicated the trap-dependent and trap-independent inactivations are both thiol dependent processes (e.g. S-nitrosation). However, the inactivation rate with GSH was not zero since 10 mM GSH is not saturating for nitrosothiol reduction (the apparent K_m value for GSH is 13 mM; Table 1), NO binding to β2 H-NOX is reversible, other minor processes besides S-nitrosation likely also result in inactivation (e.g. protein instability). Therefore, our data is consistent with both trap-dependent and trap-independent iNOS auto-inactivation resulting from Zn²⁺-tetrathiolate S-nitrosation.

Discussion

Kinetic model of iNOS auto-inactivation

Exposure of NOS to NO lowers the activity of NOS both in vitro (8, 10, 31–33) and in cells (31, 56–58). NO is also capable of S-nitrosating the NOS Zn²⁺-tetrathiolate (7–11, 30, 59), which (at least for iNOS) results in zinc loss followed by irreversible dimer dissociation and inactivation (7, 8). Here, we present a detailed kinetic model of NOS S-nitrosation and inactivation by treating arginine as a suicide substrate (Figure 2). This kinetic model has allowed for the direct and quantitative determination of the inactivation rate (λ), the NO concentration formed at infinite time ([NO]_∞), and the partition ratio between NO release and NOS S-nitrosation/inactivation. As discussed below, perturbation of this kinetic model by varying the concentration of NO traps and reductants provided insight into the role of NOS S-nitrosation in NOS auto-inactivation, the mechanism of NOS S-nitrosation, and the potential role of NOS S-nitrosation in the initial formation of nitrosothiols such as GSNO.

S-nitrosation and iNOS auto-inactivation

The majority of the evidence previously reported and expanded upon here points to S-nitrosation of the Zn²⁺-tetrathiolate as the primary mechanism of NOS auto-inactivation, although some alternative mechanisms have been described including modification of the pterin cofactor (see Supporting Information). The timescale of iNOS S-nitrosation (Figure 1A) correlated exactly with the observed loss in iNOS activity (Figure 1B). Furthermore, the ability of reducing agents (i.e. GSH and TCEP) to protect iNOS from auto-inactivation (Figure 4) also directly correlated with a decrease in iNOS S-nitrosation (Figure 5). Previous studies showed that treatment of activated macrophages with GSNO or S-nitroso-N-acetyl-penicillamine (SNAP) inactivated iNOS in a concentration-dependent manner, consistent with inactivation occurring through transnitrosation of the Zn²⁺-tetrathiolate (56). SNAP addition also inhibited nNOS activity in cytosolic extracts from rat brain (56). Furthermore, SNAP inhibition of nNOS was irreversible consistent with nNOS dimer dissociation following S-nitrosation of the Zn²⁺-tetrathiolate (56) as the cause of inactivation.

Mechanism of iNOS S-nitrosation

Having determined that iNOS S-nitrosation is the primary mechanism of iNOS auto-inactivation, we sought insight into the mechanism of iNOS S-nitrosation. As mentioned above, S-nitrosation is not a simple addition of NO to thiols; the conversion of a thiol to a nitrosothiol involves a 1-electron oxidation. There are two main mechanisms of S-nitrosation (60). An aerobic mechanism (with O₂ as the oxidant, forming N₂O₃ as the nitrosating agent) in which N₂O₃ reacts with free thiols to form nitrosothiols at a rate of ~6.6 × 10⁷ M⁻¹s⁻¹ (61). Alternately, thiyl radicals of cellular thiols (e.g. GS•) can react with NO at a rate of ~3 × 10⁹ M⁻¹s⁻¹ (62) to produce nitrosothiols. For iNOS, O₂ appears to be the oxidant for S-nitrosation, as treatment of iNOS with exogenous NO under anaerobic conditions yielded no S-nitrosation (8). This requirement of O₂ for S-nitrosation is consistent with N₂O₃ as the nitrosating agent for iNOS, but other aerobic oxidation mechanisms cannot be ruled out.

In cells, NO concentrations are in the nanomolar to low micromolar range, but the concentrations of species that bind, react with, and destroy NO are several orders of magnitude higher (heme proteins, reductants, oxygen, superoxide, iron-sulfur clusters, etc.). Lim et al. estimated using a kinetic model that, under NO concentrations representative of an inflammatory response (1 μM), solution N₂O₃ concentrations are limited to the femtomolar range (63). These low estimated N₂O₃ concentrations were primarily due to the ability of GSH to scavenge NO and react with N₂O₃. Regardless of the exact mechanism of Zn²⁺-tetrathiolate S-nitrosation, the results presented here are consistent with iNOS forming the relevant nitrosating agent sequestered within the protein environment, thus avoiding reactions with GSH and other species that destroy NO.

The trap-dependent and trap-independent components observed upon fitting our kinetic model (Figure 2) at varying trap concentrations (Figure 6) support the hypothesis that NO sequestered within the protein environment is responsible for iNOS S-nitrosation. The trap-independent component suggests that NO generated at the heme cofactor can S-nitrosate iNOS without being released into solution. Importantly, assays varying the β2 H-NOX concentration in the presence of 10 mM GSH (Figure 6B) showed that the majority of the trap-independent component was thiol-dependent consistent with inactivation due to S-nitrosation. Interestingly, examination of iNOS crystal structures revealed a tunnel between the two heme-binding sites that passes directly by the Zn²⁺-tetrathiolate (Figure 7A). Similar tunnels were also found in eNOS (pdb 1DOC) and nNOS (pdb 1OM4) crystal structures. It is important to note that the Zn²⁺-tetrathiolate is positioned directly between this tunnel and the solvent exposed surface of NOS. Therefore, S-nitrosation of the Zn²⁺-tetrathiolate might occur via this conduit tunnel resulting in surface exposed nitrosothiols that may participate in downstream transnitrosation reactions with GSH or other proteins. The hypothesis that the NOS nitrosothiols are surface exposed is supported by the ability of both GSH and TCEP (which is structurally unrelated to GSH) to efficiently reduce iNOS S-nitrosation (Figure 5). This use of a protein tunnel represents a novel mechanism to direct NO reactivity.

Figure 7.

(A) Potential tunnel for NO diffusion from the heme to the Zn²⁺-tetrathiolate within iNOS. The tunnel within a structure of the iNOS heme domain (pdb 1DWV) (85) was generated using CAVER (86). (B) Summary model of iNOS auto-inactivation through S-nitrosation of the Zn²⁺-tetrathiolate and protection from inactivation by reductants. The illustrated scheme is superimposed on the kinetic model that was developed by treating the substrate arginine as a mechanism-based inhibitor. Rescue by cellular reductants (GSH) is also shown as well as trapping of released NO by MbO₂.

The trap-dependent component suggests that a component of iNOS inactivation involves competition of the Zn²⁺-tetrathiolate and MbO₂ for solution NO. In the kinetic model (Figure 2), the rate of NOS competition for solution NO is represented by the rate constant k₆[NO] (Table 1). From the rate of NO rebinding to the heme upon photodissociation, Slama-Schwok et al. estimated that ~4 non-heme bound NO molecules can reside within the eNOS oxidase domain (64). If we estimate that, like eNOS, iNOS also possesses 4 non-heme NO binding sites per monomer in addition to the heme binding site, then the steady-state NO concentration can be estimated as ~75 nM as 15 nM iNOS was utilized in our assays. Using this analysis, the estimated bimolecular rate of NO sequestration by iNOS (k₆ in Figure 2) is ~10¹⁰ M⁻¹s⁻¹. As N₂O₃ nitrosates GSH at a rate of 6.6 × 10⁷ M⁻¹s⁻¹ (61), either the iNOS Zn²⁺-tetrathiolate is over two orders of magnitude more reactive towards N₂O₃ compared to GSH, N₂O₃ is not the relevant nitrosating agent, or all S-nitrosation occurs through the tunnel (Figure 7A). In the last case, the trap-dependent component would result from competition of NO released into solution between reaction with MbO₂ and diffusion back into iNOS (Figure 7B). Furthermore, rates of ~10¹⁰ M⁻¹s⁻¹ are within reason for diffusion of gases into protein environments (65).

Several other lines of evidence are consistent with the ability of NOS to sequester NO and generate nitrosating agents within the protein environment. First, non-heme NO binding sites have been observed in NOS crystal structures (66). Second, HbO₂ was found much more efficient in protecting iNOS from inactivation by exogenously added NO compared to generated NO at the active site during turnover (36) suggesting that NO does not dissociate from iNOS prior to S-nitrosation of the Zn²⁺-tetrathiolate. Finally, the interior of NOS is largely hydrophobic and the S-nitrosation of proteins is accelerated in a hydrophobic environment (67). Therefore, a high local concentration of NO and oxygen sequestered within NOS may accelerate the formation of the nitrosating agent (e.g. N₂O₃) involved in NOS S-nitrosation.

GSH protection of iNOS from S-nitrosation and inactivation

In assays without added reductant iNOS is fully inactivated in ~30 minutes (Figure 1B). Millimolar GSH concentrations were sufficient to protect iNOS from S-nitrosation and inactivation, but significant iNOS S-nitrosation and inactivation was observed at micromolar GSH concentrations (Figure 5 and 6). Therefore, iNOS might only inactivate in vivo once GSH concentrations reach micromolar levels. In particular, the kinetic data presented here suggests that the rate of iNOS auto-inactivation (i.e. λ) and the total concentration of NO synthesized (i.e. [NO]_∞) are carefully controlled by the concentration of reduced cellular thiols (e.g. GSH). Additionally, proteins that may be direct targets of NOS transnitrosation (e.g. COX-2, caspase-3, or arginase 1) may protect NOS from auto-inactivation. Intriguingly, iNOS is most responsive to low millimolar concentrations of GSH, which corresponds to the GSH concentration in normal cells (1–5 mM) (17). In cases where GSH levels drop from low millimolar to high micromolar concentrations (e.g. during endotexemia (68, 69) or ischemia/reperfusion (70) in hepatocytes or during macrophage activation (71)), significant iNOS inactivation would be predicted. Indeed, in activated macrophages total glutathione concentrations (GSH and GSSG) decreased by 45% and the GSH:GSSG ratio decreased from 12:1 to 2:1 after 48 hours. This decrease in GSH levels directly correlated with a drop in NOS activity (71). Depletion of cellular GSH levels through chemical means also led to a sharp decrease in iNOS activity in induced macrophages (71, 72) or hepatocytes (46, 73) and eNOS activity in endothelial cells (74–77). Addition of GSH (46, 74) or glutathione ethyl ester (72, 78) concurrently with GSH-depleting small molecules resulted in protection from NOS inactivation. However, addition of GSH to induced macrophage cytosolic extracts failed to recover iNOS activity (72), suggesting that GSH protects iNOS from inactivation in vivo but that GSH is incapable of recovering activity once iNOS is inactivated, an observation that mirrors in vitro results reported here.

Implications for NOS S-nitrosation in the physiological generation of nitrosothiols

To gain insight into the physiological relevance of NOS S-nitrosation as an initial source of nitrosothiols, we determined the partition ratio of NO release versus iNOS S-nitrosation/inactivation (Figure 2) at varying concentrations of NO traps (Figure 6). In vivo, this partition ratio likely resembles the trap-independent component in Figure 6 as the concentrations of species that bind, react with, and destroy NO are high compared to iNOS concentrations. Therefore, NO release is likely irreversible in vivo and the partition ratio is represented by k₅/k_9,11′, which is ~22,000 (Table 1). The k_9,11′ value used in this calculation was the higher value without added reductant since GSH reversal of iNOS S-nitrosation also results in the formation of a nitrosothiol, GSNO. Importantly, the half-life of NO in vivo is on the order of 2 milliseconds to 2 seconds (79, 80) and the half-lives of nitrosothiols in plasma are ~40 minutes (81). Assuming an NO half-life of one second, then a partition ratio of ~2,400 would result in equal steady state NO and nitrosothiol concentrations, which is within an order of magnitude of the determined partition ratio of ~22,000. Since iNOS produces low micromolar NO concentrations and current estimates of cellular nitrosothiol concentrations generally fall within the nanomolar range (20), the determined partition ratio is consistent with iNOS S-nitrosation being responsible, at least in part, for the physiological generation of nitrosothiols. However, future work is needed to determine if the primary function of iNOS S-nitrosation is as a mechanism to control iNOS activity or as an initial source of nitrosothiols. Furthermore, future work is needed to determine what proteins, if any, are direct targets of iNOS transnitrosation.

While the partition ratio between NO release and iNOS S-nitrosation favors NO release for iNOS, eNOS and nNOS might favor S-nitrosation. eNOS and nNOS are more susceptible to inactivation by NO (31, 33, 36) suggesting greater S-nitrosation of the constitutive NOS isoforms. In addition, S-nitrosation of eNOS and nNOS might be less coupled to dimer dissociation allowing transnitrosation reactions to occur without dimer dissociation and inactivation. Consistent with this hypothesis, S-nitrosated eNOS is mostly dimeric and both eNOS S-nitrosation and inactivation were fully reversible within endothelial cells (10). In vitro, the eNOS oxidase domain forms the most stable dimer, followed by nNOS and then iNOS (82). However, further studies are needed to determine the partition ratio between NO release and S-nitrosation/inactivation for the constitutive NOS isoforms.

As intracellular GSH concentrations range from 0.5 mM to 10 mM (17), GSNO is likely a major product of transnitrosation reactions with S-nitrosated NOS. NOS has been shown to produce nanomolar to low micromolar GSNO concentrations both in vitro (83), in cells (19), and plasma (20, 83, 84). Based on the results in Figure 3, an intracellular GSH concentration of ~3 mM approximately doubles [NO]_∞ and halves the iNOS auto-inactivation rate. This suggests that approximately equal GSNO and S-nitrosated iNOS concentrations are formed at 3 mM GSH. The balance between GSNO formation and iNOS S-nitrosation will favor iNOS S-nitrosation if GSH levels are decreased and favor GSNO if GSH levels are increased. Thus, protein transnitrosation reactions with S-nitrosated iNOS or GSNO might directly respond to GSH levels as well as the cellular redox state (Figure 7B).

In summary, iNOS S-nitrosation plays an important role in the control of NO concentrations and potentially as an initial source of nitrosothiols in vivo. Whether NOS S-nitrosation initiates protein transnitrosation signaling pathways analogous to phosphoryl transfer (16) is an interesting avenue for future work. A complete understanding of the molecular details involved in the initial formation and transfer of nitrosothiols by NOS might lead to treatments of a broad spectrum of diseases including cancer, diabetes, and other cardiovascular, pulmonary, and neurological disorders (4).

Abbreviations

cGMP

cyclic guanosine monophosphate

GSH

glutathione

GSNO

S-nitrosoglutathione

β2

beta 2 subunit of soluble guanylate cyclase

H₄B

tetrahydrobiopterin

H-NOX

Heme Nitric oxide and/or OXygen binding domain

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HbO₂

oxyhemoglobin

Mb

myoglobin

MbO₂

oxymyoglobin

metMb

metmyoglobin

NADPH

β-Nicotinamide adenine dinucleotide 2′-phosphate reduced form

NO

nitric oxide

NOS

nitric oxide synthase

eNOS

endothelial nitric oxide synthase

iNOS

inducible nitric oxide synthase

nNOS

neuronal nitric oxide synthase

sGC

soluble guanylate cyclase

SNAP

S-nitroso-N-acetyl-penicillamine

SNO

S-nitrosothiol

SOD

superoxide dismutase

TCEP

tris(2-carboxyethyl)phosphine