Biochemical Journal

Research article

Cytochrome c-mediated formation of S-nitrosothiol in cells

Katarzyna A. Broniowska , Agnes Keszler , Swati Basu , Daniel B. Kim-Shapiro , Neil Hogg

Abstract

S-nitrosothiols are products of nitric oxide (NO) metabolism that have been implicated in a plethora of signalling processes. However, mechanisms of S-nitrosothiol formation in biological systems are uncertain, and no efficient protein-mediated process has been identified. Recently, we observed that ferric cytochrome c can promote S-nitrosoglutathione formation from NO and glutathione by acting as an electron acceptor under anaerobic conditions. In the present study, we show that this mechanism is also robust under oxygenated conditions, that cytochrome c can promote protein S-nitrosation via a transnitrosation reaction and that cell lysate depleted of cytochrome c exhibits a lower capacity to synthesize S-nitrosothiols. Importantly, we also demonstrate that this mechanism is functional in living cells. Lower S-nitrosothiol synthesis activity, from donor and nitric oxide synthase-generated NO, was found in cytochrome c-deficient mouse embryonic cells as compared with wild-type controls. Taken together, these data point to cytochrome c as a biological mediator of protein S-nitrosation in cells. This is the most efficient and concerted mechanism of S-nitrosothiol formation reported so far.

  • cytochrome c
  • glutathione
  • nitrosylation
  • S-nitrosation
  • S-nitrosoglutathione
  • S-nitrosothiol

INTRODUCTION

The S-nitrosation of cellular proteins by nitric oxide (NO)-dependent processes has been widely recognized as an important post-translational modification involved in cellular signal transduction [13]. However, mechanisms of S-nitrosation in biological systems are poorly understood. Although NO can be easily oxidized to nitrogen dioxide and dinitrogen trioxide (both implicated in mechanisms of S-nitrosation [46]), at high levels of NO and oxygen, the third-order kinetics of this reaction limit or even preclude its involvement under biologically relevant conditions [7]. It has been suggested that the reaction between NO and oxygen is enhanced in hydrophobic environments due to local concentration effects [8,9], but there is little evidence that this effect is important in vivo. The reaction of NO with thiyl radical has been reported by some authors [4,6], but not others [10], to form S-nitrosothiols, but again the relevance of this process in any meaningful biological system has not been established. There has been significant interest in the role of metal ions and metalloproteins in S-nitrosothiol formation [11]; peroxidases and haemoglobin [12,13], as well as dinitrosyl iron complexes [14,15], have all been invoked as intermediates or promoters of nitrosation. Gow et al. [16] proposed that electron acceptors could facilitate S-nitrosation by oxidizing an intermediate thionitroxyl radical, formed from the addition of NO to a thiol, suggesting that single electron acceptors may facilitate S-nitrosothiol formation. We have recently observed that ferric cytochrome c, under anaerobic conditions, can efficiently promote glutathione S-nitrosation by acting as an electron acceptor [17]. The mechanism appears to involve the initial weak binding of glutathione to cytochrome c, followed by reaction with NO to generate ferrous cytochrome c and GSNO (S-nitrosoglutathione). This mechanism would become catalytic if cytochrome c is subsequently re-oxidized to the ferric form. This reaction is highly efficient with over 50% of NO converted into GSNO.

In the present study, we have further examined the role of cytochrome c in facilitating S-nitrosothiol formation in purified protein samples and in cellular systems. We show that cytochrome c facilitates S-nitrosation in both the absence and presence of oxygen. Additionally, cytochrome c can promote the S-nitrosation of purified proteins in the presence of glutathione and can also increase S-nitrosation in cell lysate. Immuno-depletion of cytochrome c from lysate results in a decrease in S-nitrosothiol formation. In addition, embryonic stem cells that lack cytochrome c have significantly lower S-nitrosothiol-generating capacity than wild-type controls when they are exposed to either NO-donor or NO-producing macrophages. Finally, antimycin A, an inhibitor of mitochondrial electron transport that enhances the level of ferric cytochrome c, increased S-nitrosothiol formation in murine macrophages stimulated with LPS (lipopolysaccharide). Similarly, treatment with NO in the presence of antimycin A led to elevated S-nitrosation in wild-type, but not in cytochrome c-deficient, cells. Taken together, these data provide evidence that cytochrome c may be an important cellular mediator of protein S-nitrosation.

MATERIALS AND METHODS

Materials

NO donors were purchased from Cayman Chemicals; all other materials were obtained from Sigma–Aldrich unless otherwise noted. All experiments were carried out using cytochrome c that was purified without trichloroacetic acid precipitation step (catalogue number C7752). Purified proteins were used as supplied, without further treatment or refining, and prepared in phosphate buffer (100 mM, pH 7.4) containing DTPA (diethylenetriaminepenta-acetic acid; 100 μM) and EDTA (100 μM).

Anaerobic experiments

Anaerobic experiments were performed using a Coy anaerobic chamber under an atmosphere of 95% nitrogen and 5% hydrogen. Buffers were equilibrated overnight inside the chamber, and other solutions were stirred within the chamber for 2 h before the start of experiments to ensure complete anaerobiosis.

MLR (multilinear regression analysis)

Absorption spectra were recorded between 450 and 700 nm every 10 s in a 1-cm-pathlength cuvette with an Agilent 8453 UV–visible spectrophotometer. Deconvolution of spectra into individual species was accomplished with MLR, using a set of pure spectra of all components as a basis. The pure components used were those shown in [17].

Cell culture and treatments

RAW 264.7 cells were cultured in DMEM (Dulbecco's modified Eagle's medium; Invitrogen) supplemented with streptomycin (200 μg/ml), penicillin (200 units/ml) and 10% (v/v) FBS (fetal bovine serum; Invitrogen). Murine embryonic cells lacking cytochrome c were a gift from Dr M. Celeste Simon (University of Pennsylvania, Philadelphia, PA, U.S.A.) and were maintained in DMEM with 4.5 g glucose/ml, 20% FBS, 25 mM Hepes, 2 mM glutamine, penicillin (100 units/ml), streptomycin (100 μg/ml), 0.1 mM MEM non-essential amino acids, 50 μM 2-mercaptoethanol (Cell and Molecular Technologies), 500–1000 units/ml mouse leukaemia inhibitory factor (Chemicon ESGRO), 2 mM sodium pyruvate and 50 μg/ml uridine as described previously [18]. Cells were grown on gelatin-covered six-well plates. Control mouse embryonic cells were cultured under similar conditions, but in the absence of additional pyruvate, uridine and Hepes. Medium without 2-mercaptoethanol and leukaemia inhibitory factor was used upon the exposure of mouse embryonic cells to LPS-stimulated RAW 264.7 macrophages.

S-nitrosation in cell lysates

Cells were lysed in lysis buffer (20 mM Tris/HCl, pH 8.0, 137 mM NaCl, 1 mM DTPA, 10% glycerol, 1% Triton X-100 and 1% protease inhibitor cocktail) and incubated with an NO donor under anaerobic or aerobic conditions for 30 min in the presence or absence of ferric cytochrome c. To prevent any subsequent S-nitrosation reactions, free thiols were blocked with 10 mM NEM (N-ethylmaleimide). For anaerobic experiments, the lysates were incubated under anaerobic conditions for 2 h before the treatment.

S-nitrosothiol determination

The S-nitrosothiol levels were determined using the tri-iodide-dependent ozone-based chemiluminescence method using a Sievers model 280 NO analyser as described previously [19,20]. Briefly, the reaction solution was made fresh daily from potassium iodide (28 mg) and I2 (18 mg) in acetic acid (3.75 ml) and double-distilled water (1.25 ml). This solution was added into the reaction vessel together with an antifoaming agent and maintained at 30°C. Samples were pre-treated with 10% (v/v) sulfanilamide (100 mM in 2 M HCl) to remove nitrite. Mercuric chloride (5 mM for 10 min) was used to verify the presence of S-nitrosothiols. A standard curve was generated based on the detector response to GSNO.

Nitrite measurements

The nitrite level in the medium was measured by the Griess assay [21]. Briefly, 200 μl of the sample was mixed with 10 μl of sulfanilamide (30 mM in 2 M HCl), followed by 10 μl of N-(1-naphthyl)ethylenediamine dihydrochloride (30 mM in 0.1 M HCl). The attenuance was measured at 540 nm and compared with a standard curve generated using sodium nitrite.

Cytochrome c immunodepletion

Protein A/G beads were coated with anti-cytochrome c antibody (BD Biosciences) or isotype-matched control IgG (Sigma) for 2 h at 4°C, and then beads were washed to remove unbound antibody. Cell lysate (450 μg) was incubated with antibody-coated beads for 3 h at 4°C, followed by centrifugation. The resulting supernatants were incubated under anaerobic conditions with Proli/NO [1-(hydroxyl-NNO-azoxy)-L-proline (ProliNONOate)] in the presence or absence of cytochrome c.

Determination of cytochrome c levels

The protein levels of cytochrome c and β-actin were probed using Western blot analysis after reducing SDS/PAGE. Briefly, the cells were harvested in lysis buffer, and cellular proteins were separated by SDS/PAGE (4–20% gel). The levels of cytochrome c and β-actin were detected using specific antibodies (MitoSciences and Sigma respectively) and visualized with enhanced chemiluminescence.

Statistics

All data are means±S.E.M. unless otherwise indicated. Statistical analysis of data was carried out using a Student's t test. Changes were considered statistically significant when P<0.05.

RESULTS

Cytochrome c-mediated S-nitrosation of GSH under aerobic conditions

In our previous study, we demonstrated that cytochrome c could efficiently promote the S-nitrosation of GSH by NO under anaerobic conditions and also in the presence of 1% oxygen [17]. In the present study, we have examined the efficiency of cytochrome c-mediated S-nitrosation under fully aerobic conditions. The addition of Proli/NO (100 μM) to GSH under aerobic conditions leads to the formation of approximately 20 μM GSNO (Figure 1A). In the presence of cytochrome c, the levels of GSNO formed in this system increased approximately 2 times (Figure 1A), indicating that, even in the presence of high concentrations of both NO and oxygen, cytochrome c-mediated GSNO formation is still competitive with NO oxidation. Figure 1(B) shows kinetic traces of the reaction between glutathione, ferric cytochrome c and NO. As one can observe, ferric cytochrome c rapidly transforms into a ferric nitrosyl form, which decays over time, with concomitant generation of ferrous cytochrome c. Under the same conditions, either GSH or Proli/NO alone reduces ferric cytochrome c much more slowly and to a lesser extent than when GSH and Proli/NO were both present (results not shown). This is in agreement with our previous studies under anaerobic conditions [17].

Figure 1 Cytochrome c-mediated S-nitrosation of glutathione with Proli/NO under aerobic conditions

(A) Glutathione (1 mM) was incubated with Proli/NO (100 μM) without or with cytochrome c (100 μM) under aerobic conditions for 30 min, followed by blocking of free thiol with excess NEM. Cytochrome c was separated on a 10 kDa filter, and GSNO was quantified in the low-molecular-mass fraction with tri-iodide-chemiluminescence. Values are means±S.E.M. (n=6). (B) Kinetic traces of the reaction between glutathione (1 mM), cytochrome c (100 μM) and Proli/NO (100 μM) under aerobic conditions. Spectra were recorded between 450 and 700 nm with a rate of 2.5 s per cycle and subjected to MLR. Continuous line: ferric cytochrome c; dotted line: ferric nitrosyl cytochrome c; dashed line: ferrous cytochrome c.

S-nitrosation of purified proteins by cytochrome c

To determine if cytochrome c could directly or indirectly facilitate the S-nitrosation of proteins, we incubated several purified proteins: HSA (human serum albumin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and aldolase, with cytochrome c and Proli/NO in the presence and absence of GSH under anaerobic conditions. Without GSH, negligible levels of S-nitrosated proteins could be detected in the presence or absence of cytochrome c (results not shown). When GSH was introduced to the system, robust cytochrome c-dependent GSNO formation and significant protein S-nitrosation were observed (Figure 2A). In this Figure, each bar represents total S-nitrosothiol and is divided into low-molecular-mass (grey) and high-molecular-mass (white) as determined by passage through a 10 kDa cut-off filter. These data suggest that protein S-nitrosation occurs via the intermediacy of GSNO as a result of transnitrosation between GSNO and the protein thiol. The efficiency of protein S-nitrosation is therefore likely to depend on the equilibrium position of the transnitrosation reaction with GSNO. When comparing the conversion of protein thiol into S-nitrosothiol, we found that HSA and aldolase were more sensitive targets to S-nitrosation than GAPDH (0.42, 0.37 and 0.26 mol/mol respectively).

Figure 2 Cytochrome c-mediated S-nitrosation of purified proteins

(A) HSA, GAPDH or aldolase (15 μM) was incubated with Proli/NO (100 μM) and GSH (1 mM) in the presence or absence of cytochrome c (100 μM) under anaerobic conditions for 30 min and then free thiol was blocked with excess NEM. Protein was separated from low-molecular-mass fraction on a 10 kDa filter, and S-nitrosothiol levels were quantified in both fractions by chemiluminescence. Open bars: protein S-nitrosothiol; shaded bars: GSNO. Values are means±S.E.M. (n=3). (B, C) HSA (100 μM) was incubated with Proli/NO (B) or with Sper/NO (C) (100 μM) in the presence or absence of cytochrome c (100 μM), and with or without GSH (1 mM) for 30 min. Free thiol was then blocked with excess NEM. Protein was separated from low-molecular-mass fraction on a 10 kDa filter, and S-nitrosothiol levels were quantified in both fractions by chemiluminescence. White bars: protein nitrosothiol; shaded bars: GSNO. Values are means±S.E.M. (n=3).

We further investigated the efficiency of S-nitrosation of HSA under anaerobic and aerobic conditions, using Proli/NO and Sper/NO {N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl-1,3-propanediamine (spermine NONOate)} [half-life: 2.3 s and 230 min at room temperature (25°C) respectively] (Figures 2B and 2C). The longer half-life of Sper/NO will create a sustained steady-state level of NO rather than an initial burst that would be generated with Proli/NO. In all cases, increased S-nitrosothiol formation was detected in the presence of GSH and cytochrome c, compared with mixtures deficient in either or both of these compounds. Under aerobic conditions, cytochrome c also promoted HSA S-nitrosation, although to a smaller extent than observed anaerobically. A similar pattern of protein S-nitrosation and GSNO formation was observed with both NO donors, although in general Sper/NO generated lower levels of S-nitrosothiols, as expected by its longer half-life of decomposition.

Cytochrome c-dependent S-nitrosation in cell lysates

To examine whether cytochrome c can facilitate protein S-nitrosation in a model biological system, cell lysate obtained from RAW 264.7 cells was incubated with NO donors in the presence or absence of cytochrome c. Under anaerobic conditions, NO, generated from Proli/NO, led to a dose-dependent increase in S-nitrosothiol levels (Figure 3A, inset). When the lysate was supplemented with ferric cytochrome c (100 μM), the S-nitrosation was greatly enhanced, achieving levels of 3.8 nmol/mg of protein (Figure 3A). Figure 3(B) shows the treatment of cell lysate with Sper/NO with and without ferric cytochrome c under aerobic conditions. Similarly, in the presence of cytochrome c, the S-nitrosation was more efficient. Sper/NO instead of Proli/NO was used under aerobic conditions due to its longer half-life to provide steady-state level of NO. Although absolute levels of S-nitrosothiols were higher under anaerobic than under aerobic conditions, ferric cytochrome c enhanced S-nitrosation in both cases.

Figure 3 Cytochrome c-mediated S-nitrosation in RAW 264.7 cell lysates

RAW 264.7 cell lysates were exposed to increasing concentrations of NO donor in the presence (closed symbols) or absence (open symbols) of cytochrome c (100 μM). (A) Samples were exposed to various concentrations of Proli/NO under anaerobic conditions in the presence and absence of cytochrome c for 30 min. Inset: cell lysate treated with Proli/NO. (B) Samples were exposed to various concentrations of Sper/NO under aerobic conditions in the presence and absence of cytochrome c for 30 min. S-nitrosothiol levels were determined by chemiluminescence. Results were normalized to protein concentration and are means±S.E.M. (n=3).

Effect of endogenous cytochrome c on S-nitrosothiol formation

To study the involvement of endogenous cytochrome c in the S-nitrosation process, cytochrome c was immunodepleted from the cell lysate. Cell lysate was equally divided and incubated with either IgG-coated Protein A/G beads or anti-cytochrome c antibody. The absence of cytochrome c in the immunodepleted lysate was confirmed by Western blotting (Figure 4A). When deoxygenated lysates were treated with Proli/NO, the levels of S-nitrosothiols were decreased by 40% in cytochrome c immunodepleted samples compared with the control (Figure 4B). To ascertain if the immunodepleted lysate was still capable of supporting S-nitrosation, exogenous ferric cytochrome c was added to these samples. S-nitrosation was increased equally in both the control and cytochrome c-depleted samples. This indicates that sample manipulation did not affect the ability of the lysates to support S-nitrosothiol formation.

Figure 4 Cytochrome c depletion

(A) Western blot analysis of immunodepleted cell lysates. (B) RAW 264.7 cell lysates were incubated with specific anti-cytochrome c antibody to deplete endogenous cytochrome c or with control IgG. Lysates were then exposed to Proli/NO (100 μM) in the presence and absence of cytochrome c (cyt c) (100 μM) under anaerobic conditions for 30 min. S-nitrosothiol levels were measured by chemiluminescence. Results were normalized to protein concentration and are means±S.E.M. (n=3, *P<0.005).

S-nitrosation in cytochrome c-deficient mouse embryonic cells

To further explore the role of cytochrome c in facilitating the S-nitrosothiol production, murine cells obtained from cytochrome c−/− embryos or from wild-type littermate embryos were treated with Sper/NO to determine whether endogenous cytochrome c plays a role in S-nitrosation in live cells. The cytochrome c-deficient status of these cells was confirmed by Western blot analysis (Figure 5A, inset). Figure 5(A) shows that cells lacking cytochrome c protein produced over 3 times lower levels of S-nitrosothiols compared with wild-type counterparts after exposure to Sper/NO.

Figure 5 Cytochrome c-mediated S-nitrosation: the effect of antimycin A

(A) Murine embryonic cells, wild-type (WT) and cytochrome c (cyt c)-null, were treated with Sper/NO (200 μM) for 1 h in HBSS (Hanks balanced salt solution) in the absence (black bars) or presence (white bars) of antimycin A (10 μM). Cells were lysed in the presence of NEM, and S-nitrosothiol levels were measured by chemiluminescence. Results were normalized to protein concentration and are means±S.E.M. (n=3,*P<0.05, **P<0.01 as compared with wild-type treated with Sper/NO). Inset: Western blot analysis of mouse embryonic cells. (B) RAW 264.7 macrophages were treated with LPS (0.5 μg/ml) in the presence of L-NAME (4 mM) for 13 h, after which the medium was replaced and cells were incubated in the presence and absence of antimycin A (10 μM) for 4 h. S-nitrosothiol levels (black bars) were measured by chemiluminescence and normalized to protein concentration. The nitrite concentration in the medium (grey bars) was measured by the Griess assay. Results are means±S.E.M. (n=3,*P<0.01).

As S-nitrosothiol synthesis requires ferric cytochrome c, we hypothesized that inhibition of mitochondrial electron transport would increase cytochrome c oxidation and therefore enhance S-nitrosothiol formation. Inhibition of mitochondrial electron transport with antimycin A significantly increased the levels of S-nitrosothiols in wild-type cells, but not in cytochrome c-null cells. These data support the role of cytochrome c in S-nitrosothiol formation; we show that S-nitrosation is less efficient in the absence of cytochrome c and that by putatively increasing the level of ferric cytochrome c, one can facilitate the S-nitrosothiol formation.

S-nitrosation from iNOS (inducible nitric oxide synthase) derived NO

We next explored S-nitrosothiol formation from iNOS-derived NO in the presence and absence of antimycin A. RAW 264.7 macrophages were treated with LPS for 13 h to induce iNOS (nitrite levels in the medium: control untreated cells, 2.3±0.1 μM; LPS, 72.6±0.9 μM). To prevent the production of NO and the formation of S-nitrosothiols during overnight induction of iNOS, an iNOS inhibitor, L-NAME (NG-nitro-L-arginine methyl ester), was added. L-NAME decreased medium nitrite levels to 5.0±0.3 μM in LPS-exposed macrophages. After iNOS induction, cells that had been treated with LPS and L-NAME were subsequently washed and cultured in L-NAME-free medium in the presence and absence of antimycin A, and cellular S-nitrosothiol formation and nitrite accumulation in the medium were monitored. Figure 5(B) shows that antimycin A did not affect nitrite accumulation during 4 h of incubation. Nevertheless, the inhibition of complex III led to an almost 2-fold increase of S-nitrosation in RAW 264.7 cells.

Finally, to directly probe the involvement of endogenous cytochrome c in S-nitrosation from iNOS-derived NO, wild-type and cytochrome c-null cells were exposed to LPS-stimulated RAW 264.7 cells. In this experiment, RAW 264.7 macrophages, cultured on transwell inserts, were stimulated with LPS in the presence of L-NAME for 12 h. Next, the cells were washed and the inserts were transferred on to plates containing mouse embryonic cells. Cells were co-cultured together for 4 h, and S-nitrosothiol levels were measured in wild-type and cytochrome c mouse embryonic cells. We detected S-nitrosothiols only in wild-type, but not cytochrome c-null, mouse embryonic cells, which were exposed to LPS-stimulated macrophages (Figure 6). Although the levels of S-nitrosothiols were close to the detection limit of the technique, peaks that were amenable to integration were observed in the wild-type cells but not in the cytochrome c-null cells. The inset of Figure 6 shows the raw traces obtained after injection of equal amounts of the wild-type and cytochrome c-null samples (~350 μg of each) into the NO analyser.

Figure 6 S-nitrosation in co-culture studies

Murine embryonic cells, wild-type (WT) and cytochrome c (cyt c)-null, were co-cultured with LPS-stimulated RAW 264.7 macrophages for 4 h. Murine embryonic cells were lysed and S-nitrosothiol levels were measured by chemiluminescence. Results were normalized to protein concentration and are means±S.E.M. (n=3). S-nitrosothiol levels were below the detection limit in cytochrome c-null cells (not detectable, N.D.). Inset: raw chemiluminescence data showing the size of the signal from wild-type and cytochrome c-null cells. Arrows indicate the injection of the sample.

DISCUSSION

In our previous study, we observed that ferric cytochrome c could act as an electron acceptor for the formation of GSNO from GSH and NO [17]. In the present study, we extend previous findings and show that this mechanism is unlikely to facilitate direct protein S-nitrosation, but is able to stimulate protein S-nitrosation through the intermediacy of GSNO. In addition, we demonstrate that immunodepletion of cytochrome c from RAW 264.7 cell lysates results in a decrease in S-nitrosothiol formation after exposure to NO. Finally, we report decreased S-nitrosothiol formation in cytochrome c-deficient cells. We proposed previously a mechanism that involved the weak binding of GSH to cytochrome c, followed by reaction of this complex with NO to form GSNO and reduced cytochrome c [17]. In many ways, this mechanism is similar to that proposed by Gow et al. [16] which invoked the one-electron oxidation of an intermediate thionitroxyl radical formed from the addition of NO to thiol by oxygen or NADP+. Although we have found no evidence that either oxygen or NAD+ [5] can act as one-electron acceptors in this reaction, ferric cytochrome c is able to act in this role. The specificity for cytochrome c may come from the fact that it has a weak binding site for GSH that facilitates this reaction [22]. However, other haem proteins, particularly haemoglobin, have been shown to support S-nitrosothiol formation, although with very low efficiency [12,13,23,24].

The presence of ferric cytochrome c results in an almost stoichiometric conversion of NO into GSNO. This is the most efficient mechanism of S-nitrosothiol formation described so far. Previous studies have identified transition metals and metalloproteins as mediators of S-nitrosation through their ability to act as electron acceptors. In particular, the plasma protein caeruloplasmin has been shown to support NO-dependent S-nitrosation in plasma [25]. In addition, the ‘free iron pool’ has been implicated in cellular S-nitrosothiol formation through the intermediate formation of dinitrosyl iron complexes [15]. Cytochrome c-dependent GSNO formation is quite strongly oxygen dependent with greater yields under anaerobic conditions. This is probably due to kinetic competition between GSH/cytochrome c and oxygen for NO. As the reaction between NO and oxygen is a second-order reaction in NO [26,27], this competition will increasingly favour reaction with oxygen as NO concentrations increase. Although the reaction of NO with oxygen generates nitrosating agents that can generate S-nitrosothiols, the efficiency is low, and the major product is thiol disulfide [4,5]. Our data indicate that, even in the presence of atmospheric oxygen concentrations, the cytochrome c/GSH reaction is still operative and increases the efficiency of thiol S-nitrosation. At physiological concentration of NO and oxygen, this difference will be significantly increased.

Cytochrome c-dependent S-nitrosothiol formation strongly suggests the intermembrane space as a locus for thiol nitrosation. The glutathione redox buffer of this compartment is more oxidizing as compared with cytosol and matrix. Hu et al. [28] have utilized redox-sensitive YFP (yellow fluorescent protein) to probe the intermembrane space in yeast and calculated the GSH/GSSG ratio of 250:1, based on the assumption that GSH concentration in this compartment does not differ from cytosol [29]. In comparison, the cytosolic and mitochondrial matrix GSH/GSSG ratio is 3000:1 and 9000:1 respectively. In agreement with these studies, an increased oxidation of the redox-sensitive GFP (green fluorescent protein) in the mitochondrial intermembrane space, as compared with cytosol, was also observed in the smooth muscle cells (intermembrane space: 47.7%, cytosol: 18.6%) [30]. Although these reports indicate more oxidizing character of the intermembrane space, they also point out that a vast majority of the glutathione pool is in the reduced state.

Experiments performed with cytochrome c-deficient cells strongly indicate that S-nitrosothiols are largely formed by a cytochrome c-dependent mechanism in a cellular environment. These cells are derived from mouse embryos at day 8.5, as cytochrome c deficiency is embryonically lethal [31]. It has been demonstrated that reintroduction of cytochrome c into these cells restores their ability to respire, indicating that they contain otherwise functional mitochondria [18]. Cells lacking cytochrome c generated significantly lower levels of S-nitrosothiols when exposed to NO donor. Moreover, S-nitrosation was observed only in wild-type cells when mouse embryonic cells were co-cultured with LPS-stimulated RAW 264.7 cells.

As the mechanism of S-nitrosation requires ferric cytochrome c, we examined whether antimycin A, an inhibitor of cytochrome c reduction from mitochondrial complex III, was able to stimulate S-nitrosation. We observed a significant increase in cytochrome c formation in the presence of antimycin A only in the wild-type cells and not in the cytochrome c-null cells. Similarly, in the case of LPS-stimulated RAW 264.7 macrophages, we detected enhanced S-nitrosation when cells were treated with antimycin A. This observation suggests that the redox state of the mitochondrial electron transport chain may be a variable in controlling the rate of cellular S-nitrosation. This control of S-nitrosation by the oxidation state of the mitochondrial electron transport chain has several intriguing possible consequences. It would be expected that NO, by inhibiting respiration at complex IV [32,33], would force cytochrome c into the ferrous state and so inhibit S-nitrosothiol formation. In contrast, the inhibition of electron flow upstream of cytochrome c by (for example) S-nitrosation or oxidation of complexes I or III would facilitate S-nitrosothiol formation. We have shown recently, in endothelial cells, that the effects of NO on cellular respiration are quite distinct from S-nitrosation [34] and are explainable solely by reversible binding of NO to complex IV. However, it is possible that in the presence of increased oxidative stress, the mitochondrial electron transfer proteins may become inhibited, leading to the promotion of S-nitrosation via a ferric cytochrome c-dependent process. We are currently investigating these speculations.

Although S-nitrosation has been celebrated as an important NO-dependent signalling paradigm, robust mechanisms of S-nitrosothiol formation in vivo have been elusive. Much emphasis has been placed on the reaction of NO with oxygen, but there is a strong argument that this reaction is not fast enough to represent a feasible mechanism of S-nitrosothiol formation in vivo. Other mechanisms discussed above involving metal ions and metalloproteins have been examined, but the fact that no efficient concerted mechanism of S-nitrosothiol synthesis in cells has been reported has been a major impediment to the concept of S-nitrosation as a signalling paradigm. In the present study, we have demonstrated that the cytochrome c-dependent formation of S-nitrosothiols that we previously observed functions in living cells and is a viable route to the S-nitrosation of cellular proteins. The functional consequences of this pathway remain to be uncovered.

AUTHOR CONTRIBUTION

Katarzyna Broniowska designed and performed experiments on cell lysates and cells and analysed the data and co-wrote the paper; Agnes Keszler designed and performed experiments on GSNO and purified proteins and analysed the data and co-wrote the paper; Swati Basu and Daniel Kim-Shapiro provided useful discussions and edited the paper before submission; and Neil Hogg designed the experiments and co-wrote the paper.

FUNDING

This study was supported by the National Institutes of Health [grant numbers GM55792 (to N.H.) and HL058091 (to D.K.-S.)].

Acknowledgments

We thank Dr M. Celeste Simon for providing mouse embryonic cells lacking cytochrome c and their wild-type counterparts. We also thank Paul Mungai for guidance in culturing mouse embryonic cells.

Abbreviations: DMEM, Dulbecco's modified Eagle's medium; DTPA, diethylenetriaminepenta-acetic acid; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSNO, S-nitrosoglutathione; HSA, human serum albumin; iNOS, inducible nitric oxide synthase; L-NAME, NG-nitro-L-arginine methyl ester; LPS, lipopolysaccharide; MLR, multilinear regression analysis; NEM, N-ethylmaleimide; Proli/NO, 1-(hydroxyl-NNO-azoxy)-L-proline (ProliNONOate); Sper/NO, N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl-1,3-propanediamine (spermine NONOate)

References

View Abstract