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Originally published In Press as doi:10.1074/jbc.M512203200 on February 14, 2006

J. Biol. Chem., Vol. 281, Issue 15, 10056-10065, April 14, 2006
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Persistent S-Nitrosation of Complex I and Other Mitochondrial Membrane Proteins by S-Nitrosothiols but Not Nitric Oxide or Peroxynitrite

IMPLICATIONS FOR THE INTERACTION OF NITRIC OXIDE WITH MITOCHONDRIA*

Christina C. Dahm{ddagger}, Kevin Moore§, and Michael P. Murphy{ddagger}1

From the {ddagger}Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge CB2 2XY, United Kingdom and §Department of Medicine, University College London, Rowland Hill Street, London NW3 2PF, United Kingdom

Received for publication, November 14, 2005 , and in revised form, February 3, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
S-Nitrosation of mitochondrial proteins has been proposed to contribute to the pathophysiological interactions of nitric oxide (NO) and its derivatives with mitochondria but has not been shown directly. Furthermore, little is known about the mechanism of formation or the fate of these putative S-nitrosothiols. Here we have determined whether mitochondrial membrane protein thiols can be S-nitrosated on exposure to free NO from 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) by interaction with S-nitrosoglutathione or S-nitroso-N-acetylpenicillamine (SNAP) and by the NO derivative peroxynitrite. S-Nitrosation of protein thiols was measured directly by chemiluminescence detection. S-Nitrosoglutathione and S-nitroso-N-acetylpenicillamine led to extensive protein thiol oxidation, with about 30% of the modified protein thiols persistently S-nitrosated. In contrast, there was no protein thiol oxidation or S-nitrosation on exposure to 3,3-bis (aminoethyl)-1-hydroxy-2-oxo-1-triazene. Peroxynitrite extensively oxidized protein thiols but produced negligible amounts of S-nitrosothiols. Therefore, mitochondrial membrane protein thiols are S-nitrosated by preformed S-nitrosothiols but not by NO or by peroxynitrite. These S-nitrosated protein thiols were readily reduced by glutathione, so S-nitrosation will only persist when the mitochondrial glutathione pool is oxidized. Respiratory chain complex I was S-nitrosated by S-nitrosothiols, consistent with it being an important target for S-nitrosation during nitrosative stress. The S-nitrosation of complex I correlated with a significant loss of activity that was reversed by thiol reductants. S-Nitrosation was also associated with increased superoxide production from complex I. These findings point to a significant role for complex I S-nitrosation and consequent dysfunction during nitrosative stress in disorders such as Parkinson disease and sepsis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
In addition to the roles of nitric oxide (NO) in regulating vascular tone, synaptic signaling, and cellular defense, its interaction with mitochondria is now recognized to be of significance (1). Because NO produced by cytoplasmic nitric-oxide synthases (NOSs)2 easily permeates phospholipid bilayers, mitochondria are exposed to varying concentrations of NO (2). There is also evidence for an isoform of neuronal NOS within mitochondria (3–5), and an isoform of endothelial NOS is associated with the mitochondrial outer membrane (6). Although the significance of these NOS activities is uncertain, it is clear that in vivo mitochondria are exposed to levels of NO that modify their activity (1).

The best understood interaction of NO with mitochondria is the reversible inhibition of cytochrome oxidase (7, 8). This inhibition by NO is competitive with O2 and is significant at physiological O2 concentrations (9). The physiological role of this inhibition is uncertain, but it may be used to divert O2 away from respiration (10). The other important, but less well understood interaction of NO with mitochondria is through reaction with thiols (1). Protein thiols and glutathione (GSH) react with NO derivatives to produce a range of products including disulfides, sulfenic, sulfinic and sulfonic acids, and S-nitrosothiols (RSNO) (11–13). The most important NO derivative in nitrosative stress is peroxynitrite (ONOO–), which forms from the reaction of NO with superoxide (14). Thiols react rapidly with ONOO– by one and two electron oxidations to produce thiyl radicals (RS·) and sulfenic acids (RSOH), respectively, both of which usually react with GSH or another protein thiol to form a disulfide but may also form higher thiol oxidation states (11, 15–19).

S-Nitrosation of protein thiols to form protein-S-nitrosothiols (PrSNO) is of particular interest because it may enable the reversible regulation of protein function as well as contributing to nitrosative stress (20–23). S-Nitrosothiols exist in vivo (24), and techniques that are thought to visualize S-nitrosated proteins suggest that some proteins are persistently S-nitrosated (25–27). Selective S-nitrosation of specific cysteine residues with functional consequences occurs on the ryanodine receptor (27), thioredoxin (28), and p21ras (29). However, the mechanism of S-nitrosothiol formation in vivo is uncertain (23). The direct reaction of NO with a thiol does not produce RSNOs (30), although intermediates formed during NO oxidation can nitrosate thiols (30) and might be produced by the selective concentration of NO and O2 within phospholipid bilayers (31). Other possible routes to RSNOs include the reaction of NO with a thiyl radical (16) and reaction of thiols with dinitrosyl iron complexes or by the reaction of ONOO– with thiols (32).

The fate of S-nitrosothiols once formed is also unclear (23). One important reaction of RSNOs is transnitrosation, by which a nitrosonium (NO+) is transferred to another thiol (30). This is likely to be of particular importance for GSH, the predominant low molecular weight thiol in cells, which can form S-nitrosoglutathione (GSNO) and transfer the NO+ onto protein thiols. Transition metals or light release NO so RSNOs can act as storage forms of NO (23), and there may be enzymatic pathways for their decay (33, 34). RSNOs also react with thiols or possibly water to lose the nitroxyl anion (NO–) and form a disulfide or a sulfenic acid, respectively (35, 36); the sulfenic acid can react with a thiol to form a disulfide. Thus, S-nitrosation of protein thiols yields three modifications: persistent S-nitrosation, formation of a sulfenic acid, or formation of a disulfide. The disulfide can be either a protein disulfide or a mixed disulfide with glutathione. However, there is still considerable uncertainty about the basic mechanisms by which PrSNOs arise in vivo, their stability, and their functional significance.

In mitochondria, prolonged exposure to NO results in peroxynitrite formation (37–41), and this leads to GSH depletion and changes that are consistent with the S-nitrosation of respiratory complex I (1, 42, 43). GSNO has been found within mitochondria (44), and the enhanced oxidation of NO by O2 within mitochondrial membranes may also contribute to S-nitrosation (45). However, although the S-nitrosation of mitochondrial proteins has been inferred, it has not been shown directly, and its mechanism of formation is uncertain. Consequently, it is of great interest to determine whether mitochondrial protein thiols can be S-nitrosated, to find out the mechanism, and to identify persistently S-nitrosated proteins. Here we set out to determine whether S-nitrosothiols formed on mitochondrial proteins thiols by quantitating the loss of thiols and the formation of S-nitrosothiols on mitochondrial membranes exposed to free NO, low molecular weight S-nitrosothiols, or peroxynitrite. We found that exposure to S-nitrosothiols or peroxynitrite led to the extensive loss of free protein thiols, but that exposure to free NO did not. Only exposure to S-nitrosothiols led to significant PrSNO formation. This protein S-nitrosation was rapidly reversed by GSH. Complex I was among the proteins S-nitrosated, and this correlated with a loss in its activity and increased superoxide formation. These findings shed light on the pathological and physiological interactions of mitochondrial protein thiols with NO and its derivatives.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Materials—All chemicals were from Sigma unless otherwise stated. (4-Iodo)butyltriphenylphosphonium iodide (IBTP) and antitriphenylphosphonium rabbit antiserum were prepared as described (46). Polyclonal antiserum against S-nitrocysteine was from AG Scientific, and a monoclonal antibody against glutathionylated proteins was from Virogen.

Preparations—Rat liver mitochondria were prepared in STE (250 mM sucrose, 5 mM Tris-HCl, 1 mM EGTA, pH 7.4) as described previously (47), and protein concentration was measured using the biuret assay with BSA as the standard (48). Mitochondrial membranes were prepared from bovine heart mitochondria (49) by disruption of the mitochondria in a blender followed by collection and washing by centrifugation (50). Previously we showed by experiments with membrane-permeant and -impermeant thiol reagents that all exposed protein thiols are accessible to the incubation medium (51). Membranes were treated with 1 mM dithiothreitol (DTT) for 10 min at 37 °C then pelleted at 10,000 x g and washed twice before incubations (51), which were at 1 mg of protein/ml in HEND buffer (25 mM HEPES, 0.1 mM EDTA, 10 µM neocuproine, 0.1 mM N,N-bis(2-bis[carboxymethyl]aminoethyl) glycine (DTPA), pH 7.7) under light excluding conditions unless stated otherwise. Complex I was prepared by solubilization of mitochondrial membranes with dodecyl-beta-D-maltoside (Anatrace, OH) followed by ion exchange chromatography and gel filtration (56), and the pure complex I was stored in buffer containing 0.1% dodecyl-beta-D-maltoside and 10% ethylene glycol at –80 °C. Oxyhemoglobin was prepared by the reduction of 3 mM bovine methemoglobin by treatment with sufficient solid sodium dithionite to complete reduction, as indicated by the color change. This was followed by desalting on a Sephadex G-25 NAP-10 column (Amersham Biosciences), then aliquots were stored at –20 °C (53). Peroxynitrite was synthesized at 5 °C by mixing acidified 0.7 M H2O2 with an equal volume of 0.6 M NaNO2 then quenching the reaction with 1.5 M NaOH (54). Excess H2O2 was degraded by reaction with MnO2 for 30 min, which was removed by filtration through a 0.4-µm Millipore filter. Freeze fractionation at –20 °C yielded a concentrated upper layer that was isolated, quantified at A302 ({epsilon}302 = 1670 M–1 cm–1 (55)), and diluted to the required concentration in 1.5 M NaOH (54). Protein concentrations were measured by the bicinchoninic acid assay using BSA as a standard (56).

Electrophoresis and Immunoblotting—For SDS-PAGE analysis, membranes were pelleted by centrifugation (10,000 x g, 5 min), separated on 12.5% acrylamide gels using a Bio-Rad Mini Protean system, and transferred to PVDF using a Bio-Rad Mini Protean transfer cell. The blot was incubated with antiserum followed by secondary antibody-horseradish peroxidase and visualized by enhanced chemiluminescence (Amersham Biosciences). For Blue Native PAGE analysis (57), mitochondrial membranes were extracted with dodecyl-beta-D-maltoside, run on a 5–12% gradient acrylamide gel using a Bio-Rad Mini Protean system, and transferred onto PVDF using a Bio-Rad Mini Protean transfer cell. Blots were probed with antitriphenylphosphonium serum (46)/anti-rabbit IgG horseradish peroxidase and visualized by enhanced chemiluminescence.

Nitric Oxide and S-Nitrosothiol Measurements—NO concentration was measured using a NO electrode (2-mm diameter; World Precision Instruments). At the end of the incubation, oxyhemoglobin (5 µM) was added to the chamber to degrade NO to NO–3 and return the NO electrode trace to base line. To quantify S-nitrosothiols we used a chemiluminescence assay that can detect low nanomolar concentrations of S-nitrosothiols (36). Free thiols were blocked with N-ethylmaleimide (NEM), and residual NO–2 was removed by reaction with sulfanilamide. Bound NO was then released by CuI/I2 and detected by gas phase chemiluminescence on reaction with O3 in a nitric oxide analyzer model 280 (NOATM; Sievers, Boulder, CO). Parallel samples were treated with HgCl2 to degrade RSNOs selectively, and the difference between samples with and without HgCl2 gave the RSNO concentration (24). For S-nitrosothiol quantitation on mitochondrial membranes, after incubation the membranes were washed 3 times and resuspended in HEND buffer supplemented with 1 mM NEM, then split into 7 x 200-µl aliquots and stored at –20 °C until RSNO analysis. For RSNO analysis, 6 aliquots were incubated with and without 10.6 mM HgCl2 for 30 min at 4 °C, and immediately before injection into the nitric oxide analyzer, 0.5% sulfanilamide in 0.1 mM HCl was added. The RSNO content was determined by subtracting results from the parallel HgCl2-treated samples and was related to the protein concentration of the 7th aliquot as determined by the bicinchoninic acid assay. Isolated complex I was incubated at 1 mg of protein/ml, dialyzed overnight in Pierce dialysis cassettes (molecular mass cut-off, 10,000 Da) against 4 liters of phosphate-buffered saline supplemented with 1 mM NEM and 100 µM DTPA with 3 buffer changes, and then split into 5 aliquots and stored at –20 °C until RSNO. Protein concentration analyses were carried out as described above. Nitrite accumulation was measured by the Griess assay using a Griess reagent kit (Invitrogen). Mitochondrial membranes were incubated with 1 mM S-nitroso-N-acetyl-penicillamine (SNAP), 1 mM GSNO, or no additions for 10 min at 37 °C, then pelleted at 10,000 x g, and the supernatant was removed for determination of nitrite according to the manufacturer's instructions.

Respiration Rate Measurements—To measure mitochondrial respiration, rat liver mitochondria (1 mg of protein/ml) were incubated in KCl buffer (120 mM KCl, 10 mM HEPES, 1 mM EGTA, pH 7.2) at 37 °C supplemented with 10 mM succinate and 4 µg/ml rotenone in a 3-ml Clark-type oxygen electrode (Rank Brothers, Bottisham, Cambridge, UK) connected to a Powerlab data acquisition system (ADInstruments). The electrode response was calibrated assuming 205 nmol of O2/ml (58). To measure the respiration of mitochondrial membranes, membranes (0.5 mg protein/ml) were treated with 1 mM SNAP or no additions for the times indicated in 1 ml of KPi medium (50 mM KPi, 1 mM EGTA, 100 µM DTPA, 100 µM neocuproine, pH 8) at 37 °C then incubated in the oxygen electrode before respiration was initiated with either 3 mM succinate in the presence of 4 µg/ml rotenone or 500 µM NADH.

Thiol Measurements—For qualitative analysis of protein thiols in mitochondrial membranes, after incubation the membranes were washed twice with HEND buffer, then incubated with 10 µM IBTP for 10 min at 37 °C, pelleted, and resuspended in loading buffer. After separation by SDS-PAGE, protein was transferred to PVDF, and IBTP binding was detected by immunoblotting using antitriphenylphosphonium serum (46). To quantitate free thiols on mitochondrial membranes after incubation and washing, membranes (1 mg of protein/ml) were pelleted and resuspended in 1.2 ml of NaPi buffer (80 mM NaH2PO4, 1 mM EDTA, pH 8), then two 500-µl aliquots were removed and reacted with and without 200 µM dithionitrobenzoic acid (DTNB) for 5 min at 37 °C. The membranes were then pelletted, and the A412 of the supernatant was measured ({epsilon}412(thionitrobenzoic acid) = 13,600 M–1 cm–1 (59)). The protein concentration of the remaining 200-µl aliquot was determined by the bicinchoninic acid assay using BSA as a standard, and this value was used to calculate the thiol content as nmol of thiol/mg of protein. To determine whether incubation of mitochondrial membranes with SNAP led to sulfenic acid formation, membranes (1 mg of protein/ml) in HEND buffer were treated with 1 mM SNAP or no additions for 10 min at 37 °C and were then incubated with and without 5 mM dimedone for 10 min at 37 °C. They were subsequently treated with 1 mM DTT for 10 min at 37 °C before determination of free thiols by the DTNB assay. To investigate whether GSNO forms a glutathionylating sulfenic acid derivative, mitochondrial membranes (1 mg of protein/ml) were treated with and without 1 mM GSNO in the presence or absence of 5 mM dimedone for 10 min at 37 °C, then the free thiol content of the membranes was determined using the DTNB assay. For Blue Native PAGE analysis, after incubation the membranes were reacted with 1 mM IBTP, extracted, run on Blue Native PAGE, transferred to PVDF and probed for IBTP binding as above.

Complex I Activity—The activity of isolated complex I was measured as the oxidation of NADH by the decrease in A340 corrected for the absorbance of the quinone at this wavelength ({epsilon}340 = 6.81 M–1 cm–1) (60). Complex I was first incubated at 1 mg of protein/ml in HEND buffer with no additions or with 1 mM SNAP or 1 mM 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA-NONOate) for 10 min at 30 °C. After this the protein was incubated for 2 min with and without 1 mM DTT. The activity of complex I was then assayed by diluting to 5 µg of protein/ml in Tris buffer (20 mM Tris-HCl, 100 µM DTPA, 10 µM neocuproine, pH 7.5) supplemented with 100 µM NADH and 0.5 mg/ml asolectin (in 1% w/v CHAPS; Fluka). After stabilization for 2 min at 32 °C, the reaction was started by the addition of 100 µM decylubiquinone, and the decrease in A340 was measured for 3 min.

Peroxynitrite and Superoxide Measurements—To measure peroxynitrite production from mitochondrial membranes, they were incubated at 1 mg of protein/ml with 1 mM DETA-NONOate in HEND buffer, then 50 µM dihydrorhodamine 123 was added with and without 250 µM NADH, and its oxidation to rhodamine was measured as an increase in fluorescence, with excitation at 500 nm and emission at 536 nm (61). The response was quantitated against a rhodamine 123 standard curve. Peroxynitrite additions were made by spotting 1 µl of a peroxynitrite solution (1–10 mM) in 1.5 M NaOH on the wall of a 1.5-ml Eppendorf tube containing 0.2–1.2 ml of sample. The reaction was then initiated through rapid mixing by vortexing the tube. Control incubations were performed with 1-µl additions of NaOH. Oxidation of free thiols on membranes by peroxynitrite was measured by the DTNB assay as above. Superoxide dismutase (SOD)-sensitive superoxide production was measured using the reaction of superoxide with coelenterazine to produce a chemiluminescent product (62). This probe is less susceptible to interaction with the respiratory chain than acetylated cytochrome c. Membranes (1 mg of protein/ml) were incubated in KPi buffer with various additions for 10 min, then washed twice and resuspended at 1 mg of protein/ml in KPi buffer. To 0.2 mg of membranes were added 2 µM coelenterazine with and without 100 units/ml Cu, Zn-SOD with 500 µM NADH or 10 mM succinate and 4 µg/ml rotenone in a final volume of 0.5 ml KPi buffer, and the chemiluminescence of duplicate incubations was recorded in triplicate for 5 s every 30 s over 5 min using a luminometer (Berthold AutoLumatPlus, PerkinElmer Life Sciences). The difference between samples with and without SOD was taken as the SOD-sensitive chemiluminescence.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Reactions of S-Nitrosothiols and NO with Mitochondrial Protein Thiols—Because most of the mitochondrial functions affected by NO are membrane-associated, we used bovine heart mitochondrial membranes to determine the effects of NO and its derivatives on mitochondrial protein thiols (51). We have previously used this system to investigate the interaction of glutathione (GSH) and glutathione disulfide (GSSG) with mitochondrial protein thiols (51). These mitochondrial membranes contain ~80–90 nmol of thiol/mg of protein, of which ~35–50 nmol of thiol/mg of protein are exposed to the solvent in native membranes and are, thus, available to react with NO and its derivatives (51). The lack of endogenous glutathione and thioredoxin greatly simplifies interpretation of the effects of NO and its derivatives on protein thiols compared with experiments on intact mitochondria (51).

To produce free NO we used DETA-NONOate, which spontaneously decomposes to release NO (t1/2 = 3400 min at pH 7.4, 22 °C). This was compared with the S-nitrosothiols SNAP and GSNO, which nitrosate thiols by direct transfer of NO+ and also release free NO. Incubating these NO donors with mitochondrial membranes in air-saturated medium led rapidly to free NO concentrations of 0.5–2 µM (Fig. 1A), which are in the range found in pathological situations in vivo (12) and were sufficient to inhibit mitochondrial cytochrome oxidase (Fig. 1B) (7). Therefore, these conditions expose mitochondrial membranes to pathophysiological NO concentrations, whereas the similar NO concentrations produced by the three donors facilitates comparison of their effects on mitochondrial thiols.

We next determined how the different NO donors affected the number of protein thiols exposed on mitochondrial membranes (Fig. 2A). There were 45–50 nmol of thiol/mg of protein exposed on native membranes, and incubation with SNAP or GSNO blocked up to 45% of these, whereas NO from DETA-NONOate had no effect (Fig. 2A). The protein thiols lost on incubation with SNAP or GSNO were restored by the thiol reductants DTT or GSH (Fig. 2B). The binding of IBTP, which labels reactive protein thiols (46, 51), was completely blocked by NEM and decreased by GSNO and SNAP but not by DETA-NONOate (Fig. 2C), confirming that reactive protein thiols were lost on incubation with SNAP/GSNO. Therefore, free NO in air-saturated medium leads to negligible protein thiol oxidation, whereas S-nitrosothiols rapidly block reactive protein thiols.


Figure 1
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  		FIGURE 1.
Interactions of NO with mitochondrial membranes and mitochondria. A, release of NO by GSNO, SNAP, and DETA-NONOate (DETA). NO release from SNAP, GSNO, and DETA-NONOate at the indicated concentrations was measured using a NO electrode in HEND buffer in the presence of 1 mg protein/ml mitochondrial membranes. Oxyhemoglobin (OxyHb, 5 µM) was added after 10 min to degrade NO to NO–3 and return the trace to base line. Concentrations of SNAP and GSNO of 10 and 100 µM released no measurable NO (data not shown). Traces are typical results of experiments repeated on at least two occasions. B, inhibition of mitochondrial respiration by SNAP, GSNO, and DETA-NONOate. Rat liver mitochondria (0.5 mg of protein/ml) were incubated in KCl medium in an oxygen electrode chamber in the presence of SNAP, GSNO, and DETA-NONOate at the concentrations indicated. SNAP and GSNO concentrations of ≤100 µM caused negligible inhibition of respiration. Traces are typical results of experiments repeated on at least two occasions.

 
Formation of S-Nitrosothiols on Mitochondrial Protein Thiols—We next determined whether the thiol loss on incubation of mitochondrial membranes with RSNO was due to persistent S-nitrosation of protein thiols (PrSHs) (Reaction 1).

Formula 1REACTION 1
To do this we quantitated S-nitrosothiol formation by releasing NO from S-nitrosothiols on the membranes using I2/I– followed by chemiluminescence detection of NO (24, 63–65). To ensure specificity for S-nitrosothiols, measurements were compared with parallel samples that had been treated with HgCl2 to degrade S-nitrosothiols selectively (24). Incubation with SNAP or GSNO led to the formation of 6–7 nmol of S-nitrosothiols/mg of protein (Fig. 3, A and B). In contrast, there was negligible S-nitrosothiol formation from free NO formed on incubation with DETA-NONOate (Fig. 3C). In plasma, incubation with NO did lead to some S-nitrosation (24), possibly due to the presence of factors such as ceruloplasmin, which may catalyze S-nitrosation. The formation of S-nitrosothiols on the membranes was prevented by pretreatment with the thiol alkylating reagent NEM, consistent with protein S-nitrosation (Fig. 3, A and B). The thiol reductants DTT or GSH degraded the S-nitrosothiols (Fig. 3, A and B). Therefore S-nitrosothiols, but not free NO, led to extensive S-nitrosation of mitochondrial protein thiols.

The maximum S-nitrosothiol formation (6–7 nmol/mg) was 65–70% less than the greatest thiol loss of ~20 nmol of thiol/mg of protein under these conditions (Fig. 2A). This disparity is most probably due to transient S-nitrosation, with S-nitrosated protein thiols rapidly losing a nitroxyl anion to form a protein disulfide (Reaction 2) (13, 23).

Formula 2 REACTION 2
However, other reactions could also contribute to the loss of thiols. S-Nitrosothiols can hydrolyze to form a sulfenic acid (21, 35, 48) (Reaction 3).

Formula 3REACTION 3

Formula 4REACTION 4
Protein sulfenates persist in certain circumstances, although most are unstable and rapidly rearrange to a disulfide (23, 35, 66) (Reaction 4). To see if protein sulfenates accumulated after S-nitrosation of mitochondrial thiols, we used the sulfenic acid-specific reagent dimedone (5,5-dimethyl-1,3-cyclohexanedione), which converts sulfenic acids to a stable thioether derivative that is not reduced by DTT (11). When SNAP-treated mitochondrial membranes were reacted with dimedone followed by DTT, the thiol content was the same as SNAP-treated membranes that had not been dimedone-treated (data not shown). This suggests that negligible amounts of persistent sulfenic acids arise under these conditions. Inclusion of 5 mM dimedone during the incubation of membranes with GSNO did not affect the extent of protein thiol loss (data not shown), making it unlikely that there was significant involvement of transient sulfenic acid formation on either protein thiols or GSNO species during protein thiol oxidation by S-nitrosothiols.


Figure 2
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  		FIGURE 2.
Effect of NO donors on exposed protein thiols. A, effect of NO donors on free protein thiols measured by DTNB. Mitochondrial membranes at 1 mg of protein/ml in HEND buffer were incubated with SNAP, GSNO, or DETA NONOate (DETA) at the concentrations indicated before washing. Protein thiols were then measured using DTNB. Data are the means ± S.E. of three independent experiments. *, p < 0.01; **, p < 0.005 (by Student's t test). Control indicates no additions. This thiol blockage by SNAP, GSNO, or DETA was reversed by DTT or GSH (data not shown). B, reversal of effects of NO donor reactions. Membranes at 1 mg of protein/ml in HEND buffer were treated with 1 mM SNAP, GSNO, or DETA for 10 min and then washed and treated with DTT (1 mM) or GSH (10 mM) for 10 min before measurement of thiols using DTNB. The dotted line is the level of the untreated control. Data shown are the means ± S.E. of three independent experiments. C, blocking of free thiols by NO donors. Membranes were exposed to NO donors or to 1 mM NEM as above then washed, incubated with 10 µM IBTP, and separated by SDS-PAGE, transferred to PVDF, and probed for IBTP binding. Decreased intensity corresponds to increased NO donor-dependent thiol modification. {alpha}TPP, antitriphenylphosphonium.

 


Figure 3
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  		FIGURE 3.
S-Nitrosation of membrane protein thiols. Bovine heart mitochondrial membranes at 1 mg of protein/ml were incubated in HEND buffer with SNAP (A), GSNO (B), or DETA-NONOate (DETA) (C) as indicated. Some samples were pretreated with 10 mM NEM for 10 min. After incubation with the NO donors, some samples were treated with DTT (1 mM) or GSH (10 mM) for 10 min. Samples were then split in two and treated with and without HgCl2, and RSNO concentration was quantified. Data are the means ± S.E. of three independent experiments, each determined in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (to NEM-pre-treated samples by Student's t test).

 
The accumulation of the low molecular weight disulfides GSSG/N-acetyl penicillamine disulfide on incubation of membranes with GSNO/SNAP could directly oxidize protein thiols by thiol-disulfide exchange (51). However, GSSG production from GSNO is known to be minimal in the absence of light or Cu(I) (67). To see if this was also the case in our experiments, we estimated the maximum possible accumulation of low molecular weight disulfides during our incubations. To do this we measured the amount of GSNO or SNAP that decayed during a 10-min incubation with membranes, which corresponds to the sum of the PrSNO and NO –2 formed. Using the Griess assay we found that incubation of 1 mM SNAP or GSNO with membranes led to formation of 6.5 ± 0.9 and 3.0 ± 0.5 µM NO –2, respectively. Adding these values to the maximum [PrSNO] of 7 µM indicates that at most 14 µM S-nitrosothiol decayed during the incubation, corresponding to a maximum possible disulfide concentration of 7 µM. To see if such a disulfide concentration could oxidize protein thiols, we incubated mitochondrial membranes with 10 µM GSSG for 10 min and found that this did not affect the protein thiol content (data not shown). Therefore, the accumulation of low molecular weight disulfides does not contribute to protein thiol oxidation in our experiments.


Figure 4
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  		FIGURE 4.
Predominantly intraprotein disulfide bond formation on SNAP-treated mitochondrial membrane proteins. Bovine heart mitochondrial membranes at 1 mg of protein/ml were incubated in HEND buffer with 1 mM SNAP for 10 min. After incubation, the samples were resuspended in SDS-PAGE loading buffer supplemented with 50 mM NEM or with 100 mM DTT. Proteins were separated by SDS-PAGE and visualized by Coomassie staining.

 
Finally, there is the possibility of a direct reaction between a protein thiol and an S-nitrosothiol to displace NO–, leaving a mixed disulfide (PrSSR; Reaction 5) that could either persist or rearrange to form an internal disulfide (23).

Formula 5REACTION 5
However, fresh solutions of GSNO are known to be poor glutathionylating agents (68). To see if this was also the case under our conditions, we measured the relative extents of glutathionylation of mitochondrial membranes by GSNO or GSSG using an antibody against glutathionylated proteins (51). This confirmed that GSNO was far less effective at promoting protein glutathionylation than GSSG (data not shown). Therefore, it is unlikely that Reaction 5 contributes to protein thiol oxidation, but if it does then there is minimal accumulation of a glutathionylated protein intermediate, and the product is an intraprotein disulfide.

The nature of the protein disulfides was explored by separating out proteins from S-nitrosothiol-treated membranes by non-reducing SDS-PAGE in the presence of NEM to prevent the formation of artifactual protein-protein cross-links during denaturation in SDS (51) (Fig. 4). This showed that the migration of the vast majority of the abundant proteins was unaffected by treatment with SNAP, and comparison with the DTT-treated sample indicated that there were minimal interprotein cross-links between abundant proteins. This is similar to the situation of mitochondrial membranes exposed to GSSG, where there are also minimal interprotein cross-links (51) and suggests that the majority of disulfides formed on abundant proteins after S-nitrosation are intraprotein cross-links. However, we cannot exclude the possibility that a small number of inter-protein disulfide bonds form.

To summarize, the predominant reaction products formed when mitochondrial protein thiols are exposed to S-nitrosothiols are persistent S-nitrosated protein thiols and protein disulfides. The protein disulfides formed in this system are largely intraprotein disulfides, and the predominant route to their formation is probably the rearrangement of a transient S-nitrosated protein to a disulfide by Reaction 2. This is similar to the situation when mitochondrial membranes were exposed to GSSG (51). Under those conditions there was extensive loss of protein thiols. Although some of this thiol loss was due to persistently glutathionylated protein thiols, most was due to the accumulation of disulfides formed by transiently glutathionylated proteins rearranging to a disulfide by the displacement of GSH.

Interaction of Peroxynitrite with Mitochondrial Protein Thiols—Because peroxynitrite (ONOO–) reacts rapidly with thiols and is formed within mitochondria from the reaction of NO with superoxide from the respiratory chain (52, 53), we next investigated its reactivity with mitochondrial protein thiols. Bolus additions of ONOO– led to a concentration-dependent loss of thiols that was reversed by DTT but not by GSH (Fig. 5A). This contrasts with the effect of S-nitrosothiols, which were fully reduced by both DTT and GSH (Fig. 2B), and suggests that ONOO– may produce sulfinic acids, which can be reduced by DTT but not by GSH (68). It has been proposed that ONOO– reacts directly with protein thiols to form S-nitrosothiols (Reaction 6) (32).

Formula 6REACTION 6
To see if this was possible, we measured the accumulation of S-nitrosothiols on membranes exposed to a bolus of ONOO– (Fig. 5B) and found negligible S-nitrosothiol formation, accounting for at most 2% of the thiols lost on exposure to ONOO–.

It is also possible that ONOO– produced by the interaction of NO with mitochondrially produced superoxide (Formula 6) might form protein thiyl radicals that then react with NO to form S-nitrosothiols (Reaction 7).

Formula 7REACTION 7
To see if this was the case, we incubated mitochondrial membranes with DETA-NONOate and the respiratory substrate NADH (Fig. 5, C and D). Under these conditions the membranes produced superoxide that reacted with NO to form ONOO–, as indicated by the SOD-sensitive NO consumption and the oxidation of dihydrorhodamine 123 (61) (data not shown). This ONOO– production led to a small but significant loss of free thiols (Fig. 5C); however, the amount of PrSNO accumulation was again negligible (Fig. 5D). Therefore, exposure of mitochondrial protein thiols to ONOO– leads to thiol oxidation but not to protein S-nitrosation.


Figure 5
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  		FIGURE 5.
Thiol oxidation and S-nitrosothiol formation on exposure to peroxynitrite. A, effect on thiols of peroxynitrite addition. Membranes at 1 mg of protein/ml in HEND buffer were treated with peroxynitrite and then with DTT or GSH, after which the free thiol content was determined. Data are the mean ± S.E. of three independent experiments. B, effect of ONOO– on RSNO formation in membranes. Membranes at 1 mg of protein/ml in HEND buffer were treated with 1 mM NEM or no additions, then treated with ONOO– and GSH or DTT as indicated and washed, and RSNO concentration was determined. Data are the means ± S.E. of three independent experiments. C, effect on exposed thiols of respiration in the presence of DETA-NONOate. Membranes (1 mg of protein/ml) in HEND buffer were incubated with 1 mM DETA NONOate for 10 min before the addition of 10 mM NADH and incubation for a further 10 min. Then the samples were treated with the reagents as indicated and washed, and free thiols were quantitated using DTNB. Data are the means ± S.E. of three independent experiments. *, p < 0.005 by Student's t test. D, effect of 1 mM DETA-NONOate on RSNO formation in respiring mitochondrial membranes. Membranes at 1 mg of protein/ml in HEND buffer were treated with 1 mM NEM or no additions then with 1 mM DETA-NONOate and 10 mM NADH followed by 10 mM GSH, 1 mM DTT, or no additions as indicated. Samples were then washed, and RSNO content was quantified. Data are the means ± S.D. or range of three independent experiments.

 


Figure 6
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  		FIGURE 6.
S-Nitrosation of complex I. A, S-nitrosation of isolated complex I in response to NO donors. Isolated complex I at 1 mg of protein/ml in HEND buffer was incubated with 1 mM SNAP, 1 mM GSNO, 1 mM DETA-NONOate (DETA), or no additions as indicated. Some samples were pretreated with 10 mM NEM for 10 min, and the RSNO content was determined. Data are the means ± S.E. of three independent experiments, each determined in triplicate. B, S-nitrosation of isolated complex I on reaction with peroxynitrite. Bovine heart complex I (1 mg protein/ml) was incubated with 1 mM DETA-NONOate in the presence of NADH and reacted with 250 µM peroxynitrite (ONOO–) or no additions, then RSNO formation was assessed. Data are means ± S.E. of three independent experiments, each determined in triplicate. *, p < 0.05, by Student's t test.

 
Identification of Complex I as an S-Nitrosated Protein—It is of considerable interest to identify mitochondrial protein thiols that become persistently S-nitrosated on exposure to low molecular weight nitrosothiols. S-Nitrosated proteins have been detected using specific antibodies (26), but these antibodies do not work on immunoblots, as we were unable to detect S-nitrosated BSA on dot blots using this procedure (data not shown). The biotin switch technique (25), which has also been used to identify S-nitrosated proteins (25, 28, 69), works by blocking unreacted thiols, after which S-nitrosothiols are selectively reduced with ascorbate, reacted with a biotinylated thiol reagent, and detected on immunoblots. However, in our hands ascorbate reduction generated false positives by reducing disulfides formed on GSSG-treated mitochondrial membranes (data not shown), consistent with a similar limitation reported by others (70). Consequently, it was unclear if the proteins biotinylated after exposure to an NO donor originally were S-nitrosothiols or disulfides. Therefore, we instead adopted a candidate protein approach to investigate potential S-nitrosated mitochondrial thiol proteins (27).


Figure 7
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  		FIGURE 7.
Effect of S-nitrosation on complex I activity and superoxide formation. A, effect of SNAP on respiration rate of mitochondrial membranes. Membranes at 1 mg of protein/ml in HEND buffer were incubated with 1 mM SNAP as indicated, then washed and incubated with (triangles) or without (squares) 1 mM DTT for a further 10 min at 37 °C. They were then pelleted and resuspended in KPi buffer, and the rates of respiration on 500 µM NADH (filled symbols) or 3 mM succinate with 4 µg of rotenone/ml (open symbols) were determined. Data are expressed as the percentage of a parallel incubation of the same duration but without SNAP. Data are the means ± S.E. of three independent experiments. B, effect of SNAP and DETA-NONOate on the activity of isolated complex I. Isolated complex I was incubated at 1 mg of protein/ml in HEND buffer with no additions or with 1 mM SNAP or 1 mM DETA-NONOate then with and without 1 mM DTT before assaying the activity of the complex. Data are the means ± S.E. of three independent determinations in duplicate. *, p < 0.01 by Student's t test. C, effect of SNAP on superoxide production by mitochondrial membranes. Membranes at 1 mg of protein/ml in KPi buffer were treated with no additions or with 1 mM SNAP then with and without 1 mM DTT before being washed and incubated at 4 mg of protein/ml in KPi buffer with 2 µM

 
Mammalian complex I of the mitochondrial respiratory chain is a large (~980 kDa) protein of at least 46 subunits (71) and is a candidate for S-nitrosation. Complex I has exposed, reactive thiols (46, 51, 72), and nitric oxide donors induce a reversible inhibition of complex I consistent with S-nitrosation (42, 43). The implications of S-nitrosation for complex I are of particular interest because complex I is the first complex of the respiratory chain, the major source of ROS within mitochondria, and is inactivated by nitrosative stress in a number of pathologies, including sepsis and Parkinson disease (73, 74). Previously we have shown that the reactive thiols on complex I in the isolated complex or in mitochondria were blocked on incubation with SNAP (72), and we confirmed that both SNAP and GSNO also blocked reactive thiols on complex I under the conditions described for Figs. 2 and 3 (data not shown). However, it was unclear if this loss of complex I thiols was due to persistent S-nitrosation or to loss of thiols by formation of protein disulfides. Because the S-nitrosation of complex I has only been inferred from indirect measurements, we determined whether complex I could be persistently S-nitrosated by S-nitrosothiols, free NO, or by peroxynitrite. To do this we incubated isolated complex I with SNAP, GSNO, or DETA-NONOate and measured whether there was any accumulation of S-nitrosothiols (Fig. 6A). This showed that about 4 mol of PrSNO/mol of complex I were formed by SNAP or GSNO but not by DETA-NONOate (Fig. 6A). In contrast, treatment with peroxynitrite led to less than 0.1 mol of S-nitrosothiol/mol of complex I (Fig. 6B). This is the first direct demonstration that complex I can be persistently S-nitrosated and that this contributes to the loss of its protein thiols under nitrosative stress.

Effect of S-Nitrosation on Complex I Function—To understand the pathophysiological consequences of S-nitrosation of complex I, we first explored how conditions that led to S-nitrosation of mitochondrial membranes affected complex I activity (Fig. 7A). To do this we incubated mitochondrial membranes with SNAP to cause S-nitrosation. We then washed the membranes to remove SNAP and measured the rates of respiration on NADH, a complex I substrate, and on succinate, a complex II substrate. Incubation with SNAP led to a time-dependent loss of complex I-linked respiration, with nearly 60% of the activity lost after 30 min and more than 80% lost over 60 min (Fig. 7A). Complex II-linked respiration was also affected by SNAP but to a lesser degree than NADH-linked respiration over 30 min, when respiration declined by less than 40% (Fig. 7A). In contrast, incubation with 1 mM DETA-NONOate did not lead to a significant decline in complex I-linked respiration after 30 or 60 min (data not shown). The inactivation of both complex I- and complex II-linked respiration by incubations of up to 60 min was reversed by treatment with DTT (Fig. 7A); however, after 120 min of incubation the activity of complex I was not fully recovered by DTT, indicating irreversible damage. To confirm that S-nitrosation of complex I correlated with its inactivation, we incubated isolated complex I with SNAP and then measured its activity (Fig. 7B). Incubation of complex I with SNAP for 10 min led to a ~30% inactivation of complex I, whereas DETA-NONOate had no effect on activity. The inhibition by SNAP was fully reversed by DTT (Fig. 7B). Therefore, S-nitrosation is associated with time-dependent loss of activity of complex I in a similar manner to that of its incubation with thiol oxidants such as GSSG (51). The inhibition that correlates with S-nitrosation may be due to the S-nitrosation itself, to disulfide formation within complex I, or to a combination of both.

Complex I is a major source of superoxide within mitochondria, and inhibition of complex I or exposure to thiol oxidants increases superoxide production (72). To see if S-nitrosation affected superoxide production by complex I, we incubated membranes with SNAP for 10 min and then measured the production of superoxide using SOD-sensitive coelenterazine chemiluminescence. This was done using either the complex I-linked substrate NADH or the complex II-linked substrate succinate to distinguish between superoxide produced by complex I and that produced elsewhere in the respiratory chain. NADH led to an increase in superoxide production relative to membranes in the absence of respiratory substrate (Fig. 7C). However, treatment with SNAP led to a further 20% increase in superoxide production that was reversed by DTT treatment (Fig. 7C). In contrast, the complex II-linked substrate succinate led to less superoxide production than NADH, and this production was unaffected by either SNAP or DTT (Fig. 7C). Therefore S-nitrosation correlates with an increase in superoxide production by complex I.

Conclusions—We have shown that the formation of S-nitrosothiols on the exposed, reactive proteins thiols of mitochondrial membrane proteins can occur on exposure to low molecular weight S-nitrosothiols. However, S-nitrosothiol formation on exposure to free NO, even in the presence of O2, or to peroxynitrite was negligible, although peroxynitrite did lead to extensive thiol oxidation. This is the first direct demonstration that S-nitrosothiols can form on mitochondrial membrane proteins. In addition to persistent S-nitrosation, exposure to S-nitrosothiols also led to the extensive oxidation of protein thiols to intraprotein disulfides with about twice as many protein thiols forming disulfides as formed persistent S-nitrosothiols. The mechanism of the S-nitrosothiol and disulfide formation was primarily via transnitrosation from the S-nitrosothiol to the protein thiol. Some of these S-nitrosated protein thiols persisted, but the majority reacted further with an adjacent protein thiol to displace NO– and form a disulfide. Therefore, exposure of mitochondrial membranes to S-nitrosothiols leads to two main products, persistent S-nitrosothiols and protein disulfides.

In vivo, a likely pathway for S-nitrosation of mitochondrial membranes exposed to NO is via initial formation of GSNO. The accumulation of GSNO within mitochondria has been reported (44), but its origins are unclear. It could be that the direct reaction of ONOO– with GSH yields sufficient GSNO, or there could be other mechanisms for its generation, for example the direct formation of GSNO by mitochondrial NOS. Once formed, the protein S-nitrosothiols and disulfides were relatively stable in the absence of low molecular weight thiols. However, GSH rapidly reduced most protein S-nitrosothiols and disulfides back to thiols; therefore, under normal conditions in vivo most protein S-nitrosothiols will only survive transiently. This is consistent with the important role for GSH in protecting mitochondrial function in cells from nitrosative stress. However, when the glutathione pool is oxidized or depleted, protein S-nitrosation may be far more extensive, for example during Parkinson disease or septic shock (73, 75, 76). Therefore, the extent of S-nitrosation of mitochondrial protein thiols and its consequent pathological or physiological implications is critically dependent on the redox status of the mitochondrial glutathione pool, and the bulk S-nitrosation of mitochondrial proteins will only occur under conditions of nitrosative stress. The reversible formation of S-nitrosothiols is thought to be of regulatory significance; however, our findings indicate that when the glutathione pool is reduced, the majority of S-nitrosated proteins will be rapidly degraded by GSH. Even so, it is possible that a small proportion may persist or become glutathionylated, with relevance for mitochondrial regulation. Among the proteins S-nitrosated was complex I, and this S-nitrosation correlated with decreased enzymatic activity and increased superoxide formation. This confirms and extends earlier suggestions that complex I is S-nitrosated under conditions of nitrosative stress (1, 42, 43) by showing S-nitrosation directly, in contrast to those studies in which S-nitrosation was only inferred indirectly. The susceptibility of complex I to inactivation and increased superoxide production on S-nitrosation indicates that it has a significant role during nitrosative stress in pathologies such as Parkinson disease and septic shock and suggests that therapies designed to prevent S-nitrosation and protein thiol oxidation on complex I are a promising strategy.

In conclusion, we have shown that mitochondrial membrane proteins, including complex I, can be S-nitrosated by S-nitrosothiols but not NO or peroxynitrite. Extensive S-nitrosation will only persist during nitrosative stress when the mitochondrial glutathione pool is oxidized. Future work will identify the thiols affected on complex I, the mechanism of S-nitrosation in vivo, and the significance of this phenomenon for mitochondrial dysfunction.


   FOOTNOTES
 
* 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. Fax: 44-1223-252905; E-mail: mpm{at}mrc-dunn.cam.ac.uk.

2 The abbreviations used are: NOS, nitric-oxide synthase; BSA, bovine serum albumin; Complex I, NADH-ubiquinone oxidoreductase; DETA-NONOate, 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene; DTNB, 5,5'-dithiobis(2-nitrobenzoic) acid; DTPA, N,N-bis(2-bis[carboxymethyl]aminoethyl) glycine; DTT, dithiothreitol; GSNO, S-nitrosoglutathione; GSSG, glutathione disulfide; IBTP, (4-iodobutyl)triphenylphosphonium; coelenterazine with and without 100 units/ml SOD and 500 µM NADH or 10 mM succinate with 4 µg/ml rotenone. The coelenterazine chemiluminescence of each incubation was recorded in triplicate. Data are the SOD-sensitive chemiluminescence in relative light units (RLU) and are the means ± S.E. of three independent experiments, each determined in duplicate. *, p < 0.05 to membranes with NADH; **, p < 0.05 to SNAP-treated membranes with NADH (by Student's t test). NEM, N-ethylmaleimide; NO, nitric oxide; NOx, NO-derived species; ONOO–, peroxynitrite; PrSNO, protein S-nitrosothiol; PVDF, polyvinyl difluoride; RSNO, S-nitrosothiol; SNAP, S-nitroso-N-acetyl-DL-penicillamine; SOD, superoxide dismutase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Tracy Prime for expert technical assistance, Dr. Judy Hirst, Dr. Richard J. Shannon, and Steven Sherwood for samples of purified complex I, and Richard Olloson and Alireza Mani for assistance with the protein S-nitrosothiol measurements.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Moncada, S., and Erusalimsky, J. D. (2002) Nat. Rev. Mol. Cell Biol. 3, 214–220[CrossRef][Medline] [Order article via Infotrieve]
  2. Lancaster, J. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8137–8141[Abstract/Free Full Text]
  3. Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291–296[CrossRef][Medline] [Order article via Infotrieve]
  4. Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038–11043[Abstract/Free Full Text]
  5. Kanai, A. J., Pearce, L. L., Clemens, P. R., Birder, L. A., Van Bibber, M. M., Choi, S. Y., de Groat, W. C., and Peterson, J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 14126–14131[Abstract/Free Full Text]
  6. Gao, S., Chen, J., Brodsky, S. V., Huang, H., Adler, S., Lee, J. H., Dhadwal, N., Cohen-Gould, L., Gross, S. S., and Goligorsky, M. S. (2004) J. Biol. Chem. 279, 15968–15974[Abstract/Free Full Text]
  7. Brown, G. C., and Cooper, C. E. (1994) FEBS Lett. 356, 295–298[CrossRef][Medline] [Order article via Infotrieve]
  8. Cleeter, M. J. W., Cooper, J. M., Darley-Usmer, V. M., Moncada, S., and Schapira, A. H. V. (1994) FEBS Lett. 345, 50–54[CrossRef][Medline] [Order article via Infotrieve]
  9. Kindig, C. A., McDonough, P., Erickson, H. H., and Poole, D. C. (2002) Respir. Physiol. Neurobiol. 132, 169–178[CrossRef][Medline] [Order article via Infotrieve]
  10. Hagen, T., Taylor, C. T., Lam, F., and Moncada, S. (2003) Science 302, 1975–1978[Abstract/Free Full Text]
  11. Radi, R., Beckman, J. S., Bush, K. M., and Freeman, B. A. (1991) J. Biol. Chem. 266, 4244–4250[Abstract/Free Full Text]
  12. Murphy, M. P. (1999) Biochim. Biophys. Acta 1411, 401–414[Medline] [Order article via Infotrieve]
  13. Costa, N. J., Dahm, C. C., Hurrell, F., Taylor, E. R., and Murphy, M. P. (2003) Antioxid. Redox Signal. 5, 291–305[CrossRef][Medline] [Order article via Infotrieve]
  14. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1620–1624[Abstract/Free Full Text]
  15. Zhang, H., Squadrito, G. L., Uppu, R. M., Lemercier, J. N., Cueto, R., and Pryor, W. A. (1997) Arch. Biochem. Biophys. 339, 183–189[CrossRef][Medline] [Order article via Infotrieve]
  16. Karoui, H., Hogg, N., Frejaville, C., Tordo, P., and Kalyanaraman, B. (1996) J. Biol. Chem. 271, 6000–6009[Abstract/Free Full Text]
  17. Quijano, C., Alvarez, B., Gatti, R. M., Augusto, O., and Radi, R. (1997) Biochem. J. 322, 167–173[Medline] [Order article via Infotrieve]
  18. Gatti, R. M., Radi, R., and Augusto, O. (1994) FEBS Lett. 348, 287–290[CrossRef][Medline] [Order article via Infotrieve]
  19. Winterbourn, C. C. (1993) Free Radic. Biol. Med. 14, 85–90[CrossRef][Medline] [Order article via Infotrieve]
  20. Stamler, J. S., and Hausladen, A. (1998) Nat. Struct. Biol. 5, 247–249[CrossRef][Medline] [Order article via Infotrieve]
  21. Marshall, H. E., and Stamler, J. S. (2001) Biochemistry 40, 1688–1693[CrossRef][Medline] [Order article via Infotrieve]
  22. Cornwell, T. L., Ceaser, E. K., Li, J., Marrs, K. L., Darley-Usmar, V. M., and Patel, R. P. (2003) Am. J. Physiol. Cell Physiol. 284, 1516–1524
  23. Hogg, N. (2002) Annu. Rev. Pharmacol. Toxicol. 42, 585–600[Medline] [Order article via Infotrieve]
  24. Marley, R., Feelisch, M., Holt, S., and Moore, K. (2000) Free Radic. Res. 32, 1–9[Medline] [Order article via Infotrieve]
  25. Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst, P., and Snyder, S. H. (2001) Nat. Cell Biol. 3, 193–197[CrossRef][Medline] [Order article via Infotrieve]
  26. Gow, A. J., Chen, Q., Hess, D. T., Day, B. J., Ischiropoulos, H., and Stamler, J. S. (2002) J. Biol. Chem. 277, 9637–9640[Abstract/Free Full Text]
  27. Mannick, J. B., and Schonhoff, C. M. (2002) Arch. Biochem. Biophys. 408, 1–6[CrossRef][Medline] [Order article via Infotrieve]
  28. Haendeler, J., Hoffmann, J., Tischler, V., Berk, B. C., Zeiher, A. M., and Dimmeler, S. (2002) Nat. Cell Biol. 4, 743–749[CrossRef][Medline] [Order article via Infotrieve]
  29. Lander, H. M., Hajjar, D. P., Hempstead, B. L., Mirza, U. A., Chait, B. T., Campbell, S., and Quilliam, L. A. (1997) J. Biol. Chem. 272, 4323–4326[Abstract/Free Full Text]
  30. Butler, A. R., Flitney, F. W., and Wiliams, D. L. H. (1995) Trends Pharmacol. Sci. 16, 18–22[CrossRef][Medline] [Order article via Infotrieve]
  31. Liu, X., Miller, M. J. S., Joshi, M. S., Thomas, D. D., and Lancaster, J. R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2175–2179[Abstract/Free Full Text]
  32. van der Vliet, A., Hoen, P. A., Wong, P. S., Bast, A., and Cross, C. E. (1998) J. Biol. Chem. 273, 30255–30262[Abstract/Free Full Text]
  33. Liu, L., Yan, Y., Zeng, M., Zhang, J., Hanes, M. A., Ahearn, G., McMahon, T. J., Dickfeld, T., Marshall, H. E., Que, L. G., and Stamler, J. S. (2004) Cell 116, 617–628[CrossRef][Medline] [Order article via Infotrieve]
  34. Nikitovic, D., and Holmgren, A. (1996) J. Biol. Chem. 271, 19180–19185[Abstract/Free Full Text]
  35. Becker, K., Savvides, S. N., Keese, M., Schirmer, R. H., and Karplus, P. A. (1998) Nat. Struct. Biol. 5, 267–271[CrossRef][Medline] [Order article via Infotrieve]
  36. Shiva, S., Crawford, J. H., Ramachandran, A., Ceaser, E. K., Hillson, T., Brookes, P. S., Patel, R. P., and Darley-Usmar, V. M. (2004) Biochem. J. 379, 359–366[CrossRef][Medline] [Order article via Infotrieve]
  37. Lizasoain, I., Moro, M. A., Knowles, R. G., Darley-Usmar, V., and Moncada, S. (1996) Biochem. J. 314, 877–880[Medline] [Order article via Infotrieve]
  38. Packer, M. A., Porteous, C. M., and Murphy, M. P. (1996) Biochem. Mol. Biol. Int. 40, 527–534[Medline] [Order article via Infotrieve]
  39. Poderoso, J. J., Carreras, M. C., Lisdero, C., Riobo, N., Schopfer, F., and Boveris, A. (1996) Arch. Biochem. Biophys. 328, 85–92[CrossRef][Medline] [Order article via Infotrieve]
  40. Sarkela, T. M., Berthiaume, J., Elfering, S., Gybina, A. A., and Giulivi, C. (2001) J. Biol. Chem. 276, 6945–6949[Abstract/Free Full Text]
  41. Riobo, N. A., Clementi, E., Melani, M., Boveris, A., Cadenas, E., Moncada, S., and Poderoso, J. J. (2001) Biochem. J. 359, 139–145[CrossRef][Medline] [Order article via Infotrieve]
  42. Bolanos, J. P., Almeida, A., Stewart, V., Peuchan, S., Land, J. M., Clark, J. B., and Heales, S. J. R. (1997) J. Neurochem. 68, 2227–2240[Medline] [Order article via Infotrieve]
  43. Clementi, E., Brown, G. C., Feelisch, M., and Moncada, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7631–7636[Abstract/Free Full Text]
  44. Steffen, M., Sarkela, T. M., Gybina, A. A., Steele, T. W., Trasseth, N. J., Kuehl, D., and Giulivi, C. (2001) Biochem. J. 356, 395–402[CrossRef][Medline] [Order article via Infotrieve]
  45. Shiva, S., Brookes, P. S., Patel, R. P., Anderson, P. G., and Darley-Usmar, V. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 7212–7217