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Putative denitrosylase activity of Cu,Zn-superoxide dismutase
Free Radical Biology & Medicine 43 (2007) 830 – 836 www.elsevier.com/locate/freeradbiomed
Original Contribution
Putative denitrosylase activity of Cu,Zn-superoxide dismutase Ayako Okado-Matsumoto 1 , Irwin Fridovich ⁎ Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA Received 20 April 2007; revised 23 May 2007; accepted 30 May 2007 Available online 15 June 2007
Abstract The Cu,Zn-superoxide dismutase (SOD1) has been reported to exert an S-nitrosylated glutathione (GSNO) denitrosylase activity that was augmented by a familial amyotrophic lateral sclerosis (FALS)-associated mutation in this enzyme. This putative enzymatic activity as well as the spontaneous decomposition of GSNO has been reexamined. The spontaneous decomposition of GSNO exhibited several peculiarities, such as a lag phase followed by an accelerating rate plus a marked dependence on GSNO concentration, suggestive of autocatalysis, and a greater rate in polypropylene than in glass vessels. Dimedone caused a rapid increase in absorbance likely due to reaction with GSNO, followed by a slower increase possibly due to reaction with an intermediate such as glutathione sulfenic acid. SOD1 weakly increased the rate of decomposition of GSNO, but did so only when GSH was present; and FALS-associated mutant forms of SOD1 were not more active in this regard than was the wild type. Decomposed GSNO, when added to fresh GSNO, hastened its decomposition, in accord with autocatalysis, and when added to GSH, generated GSNO in accord with the presence of nitrite. A mechanism is proposed that is in accord with these observations. © 2007 Elsevier Inc. All rights reserved. Keywords: Cu,Zn-superoxide dismutase; Amyotrophic lateral sclerosis; S-nitrosylated glutathione; Denitrosylase activity; Free radicals
The Cu,Zn-superoxide dismutase (SOD1) converts superU oxide (O2 − ) into oxygen plus hydrogen peroxide (H2O2) utilizing a catalytic cycle in which the active site Cu(II) is alternately reduced and reoxidized during successive encounters U with O2 − as in Reactions (1) and (2):
U
Enzyme−CuðIIÞþ O2 YEnzyme−CuðIÞþ O2
ð1Þ
Enzyme−CuðIÞ þ O2 þ Hþ Y Enzyme−CuðIIÞþ HO 2
ð2Þ
U
The HO2− product of Reaction (2) then protonates to H2O2. The assay, as originally defined [1], utilized 10 μM ferricyto-
U
Abbreviations: SOD1, Cu,Zn-superoxide dismutase; FALS, familial amyotrophic lateral sclerosis; O2 −, superoxide; GSH, glutathione; GSNO, S-nitrosylated glutathione; GSSG, oxidized glutathione; dimedone, 5,5dimethyl-1,3-cyclohexanedione; EDTA, ethylenediaminetetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; PBS, phosphate-buffered saline; DMPO, 5,5-dimethyl-1-pyrroline N-oxide. ⁎ Corresponding author. Fax: +1 919 684 8885. E-mail address: [email protected] (I. Fridovich). 1 Current address: Department of Biochemistry, Osaka University Graduate School of Medicine, Osaka 565-0872, Japan. 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.05.038
chrome c, 50 μM xanthine, and 100 μM EDTA in 3.0 ml of 50 mM potassium phosphate at pH 7.8 at 25°C. Sufficient xanthine oxidase was added to cause ΔA550 nm of 0.025 per minute, due to the reduction of the cytochrome c by the flux of U O2 − produced by the xanthine oxidase reaction. Samples containing SOD inhibited the ΔA550 nm and 1 unit was defined as that amount which halved that ΔA550 nm. Possible interferences with this assay and means of detecting and avoiding them have been described [2]. Xanthine oxidase, during catalytic turnover, is subject to inactivation by metal contaminants in the buffer, and the EDTA prevents that inactivation [3], without interfering with the activity of SOD. In addition to its activity as a superoxide dismutase, SOD1 has been seen to exert a nonspecific peroxidase activity [4,5] that is CO2-dependent at neutral pH [6]. Another activity, as a weak GSH-dependent denitrosylase, has been reported [7]. This putative activity gained importance from the report that familial amyotrophic lateral sclerosis (FALS)-associated mutant forms of SOD1 exerted this activity to a greater degree than did the wildtype enzyme [8]. In both of these reports [7,8], chelating agents, such as EDTA, were reported to inhibit the denitrosylase activity. Because EDTA does not interfere with the dismutation activity
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of SOD1, it seems that it cannot access the active-site copper. Hence its ability to inhibit the denitrosylase activity suggests that this activity may not involve this active-site copper. This reasoning led us to examine the denitrosylase reactivity of SOD1. Herein we report that this activity is trivially weak and moreover note that there is no difference in this activity between the FALS-associated mutant and the wild-type forms of SOD1. Furthermore, apo-SOD1, H46R and H48Q that have a Cubinding site mutation, also had such denitrosylase activity, even though they did not have superoxide dismutase activity. We also note that decomposition of S-nitrosylated glutathione (GSNO) was more rapid in polypropylene than in glass vessels and that partially decomposed GSNO accelerated the decomposition of fresh GSNO and that largely decomposed GSNO reacts with GSH to regenerate GSNO. A mechanism is proposed that is in accord with these observations.
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metrically and by photolysis chemiluminescence. The decomposition of 200 μM GSNO with 2 mM GSH in the presence and absence of 10 μM SOD1 in PBS was measured as a function of time. The decomposition of 7 μM GSNO with 125 μM GSH in the presence and absence of 10 μM SOD1 in PBS was followed by photolysis chemiluminescence using a Nitrolite photolysis unit (Thermo Orion) and TEA 510 analyzer (Thermo Orion) [13]. Every 4 min for 24 min, 25 μl of reaction mixture was injected in the Nitrolite and total photolyzable NO species were measured. Superoxide dismutase activity Superoxide dismutase activities of SOD1 were measured by cytochrome c assay with 100 μM EDTA [1]. Results
Materials and methods Decomposition of GSNO at pH 7.4 Preparation of GSNO Fresh GSNO was synthesized as described by Jourd'heuil [7] with minor modification. Equal volumes of 1 M GSH in 1 N HCl and 1 M sodium nitrite in water were mixed for 30 min at room temperature in the dark. Then the solutions were neutralized with 10 N NaOH and diluted a minimum of 25-fold into phosphatebuffered saline (PBS) at pH 7.4 (Gibco) and kept on ice in dark. Decomposed GSNO was prepared by storing fresh 5 mM GSNO solution at room temperature in a glass vessel in the dark for 11 days. GSNO concentration was estimated using the molar extinction coefficients at 335 nm (586 M−1 cm−1) or 544 nm (17.2 M−1 cm−1) [9]. Preparation of wild-type and FALS-associated mutant human SOD1 Free cysteine-mutated (C6A and C111S) wild-type (WT), G93A, G93R, and E100G human SOD1 was produced and purified as described previously [10]. We expressed WT and G93A human SOD1 using a baculovirus vector in Spodoptera frugiperda (Sf9) cells. pVL1393-WTSOD1 and pVL1393G93ASOD1 were provided by Dr. E. R. Stadtman [11] and were cotransfected with linearized baculoviral DNA (PharMingen) into Sf9 cells. We then purified the SOD1 with DE52, CM52 (Whatman), and gel filtration (Ultrogel AcA54; Sigma). The concentrations of hSOD1 were estimated using the molar extinction coefficient at 265 nm (18,700 M−1 cm−1) [12], and copper content per subunit was estimated from the difference between the molar extinction coefficients at 265 and 655 nm (350 M−1 cm−1) [12].
In PBS at pH 7.4 at room temperature, GSNO exhibits a strong absorbance centered at 335 nm and a weaker band centered at 544 nm. The decomposition of GSNO could be conveniently followed spectrophotometrically and was faster when the mixture was in contact with polypropylene compared to in glass vessels. GSNO initially at 5.0 mM decomposed strikingly faster than did a solution initially at 2.5 mM. Moreover, the decomposition of the 5.0 mM solutions exhibited an initial lag followed by an accelerating rate. These characteristics are shown in Figs. 1A–1D and suggest a mechanism in which a product or an accumulated intermediate can augment the rate of consumption of GSNO. Effect of dimedone Dimedone, long used for the detection of aldehydes [14], apparently also reacts with sulfenic acids [15]. Dimedone at 5.0 mM absorbs strongly below 320 nm and, when mixed with 2.5 mM GSNO, caused a rapid increase in absorbance at 325 and 544 nm in PBS (pH 7.4), followed by a much slower increase; as shown in Figs. 2A and 2B. Tao and English reported that GSNO decomposition in the dark slowed dramatically when dimedone was present in GSNO solution in water, and an ∼10% loss of GSNO absorbance at 550 nm was observed after 51 h [15]. We reexamined the effect of dimedone in PBS (pH 7.4) instead of water because the stability of GSNO might be pH-dependent. It seems likely that the rapid increase in absorbance was due to the reaction of dimedone with GSNO in PBS (pH 7.4), whereas the slower increase was due to the reaction of dimedone with some product, or intermediate, of this GSNO decomposition.
Decomposition of GSNO Effects of GSH and SOD1 GSNO at 2.5 and 5 mM in PBS (pH 7.4) was stored at room temperature in the dark and UV/Vis spectra were measured using a UV-2501 or UV-2550 Shimadzu spectrophotometer. The denitrosylase activities of SOD1 were measured spectrophoto-
GSH added to GSNO had an insignificant effect on its rate of decomposition. However, when added in the presence of 10 μM SOD1, it increased that rate, but only when GSH was
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Fig. 1. Spontaneous decomposition of GSNO. UV/Vis spectra between 200 and 800 nm of (A) 2.5 mM and (B) 5 mM GSNO in PBS (pH 7.4) after standing at room temperature in the dark for 0–240 h (0, 72, 120, 144, and 240 are shown) in glass (G) and in polypropylene plastic (P) tubes are shown. The inset shows the absorbance between 500 and 600 nm. GSNO exhibits an absorption band centered at ∼ 335 nm and a weaker ∼ 544 nm band. These absorbances at 335 and 544 nm of (C) 2.5 mM and (D) 5 mM GSNO are plotted for 0–240 h.
in excess of GSNO. This is seen in Fig. 3A. The maximal effect of GSH occurred when GSH concentration exceeded GSNO concentration by a factor of 10, but even then the rate of decomposition of GSNO was very slow. It may well be that the requirement for excess GSH is explained by the reactive form being the thiolate anion rather than the thiol. Because the pKa of the thiol in GSH is 9.12, only a bit more than 1% would be ionized at pH 7.4. The same phenomenon was reported by Jourd'heuil et al. [7] and Johnson et al. [8]. We also found that the initial rate of GSNO decomposition in the presence of 2 mM GSH, 200 μM GSNO, and increasing concentration from 3 to 30 μM SOD1 followed saturation kinetics; however, less than 1 μM SOD1 did not significantly affect GSNO decomposition (Fig. 3B). Effect of SOD1 SOD1 has been reported to catalyze the denitrosylation of GSNO [7], and FALS-related SOD1 mutants have been stated to
exhibit more of this activity [8]. Actually, because the SOD1 has been applied at 10 μM or more, true catalytic turnover has yet to be seen. Fig. 4 presents the effects of several forms of SOD1 at 10 μM on the rate of decomposition of 200 μM GSNO in the presence of 2 mM GSH. Several things are immediately apparent. These are: (a) the rate of decomposition of GSNO is not more than doubled by these enzymes; (b) the mutant forms were not more active than the wild type; (c) the weak effect of SOD1 seen in glass vessels in not seen when the reaction occurred in polypropylene vessels; (d) replacing the two sulfhydryl-bearing cysteine residues of human SOD1 by alanine (C6A) and serine (C111S), which precludes glutathionylation and/or transnitrosation, was without effect; and (e) the copper contents, estimated from the molar extinction coefficients determined by Stansell and Deutsch [12], did not correlate with the apparent denitrosylase activity. It follows that the toxic gain of function of the FALS-related mutant forms of SOD1 cannot be a gain of denitrosylase activity, as has been claimed [8]. The spectrophotometric detection of GSNO is not as sensi-
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proportions with 0.14 mM fresh GSNO, in the presence of 1.4 mM GSH, and rates of change of absorbance at 335 nm were followed. Fig. 5 shows that an admixture of small amounts of aged GSNO with fresh GSNO plus GSH caused a decline in absorbance at 335 nm, whereas at high ratios of the aged to the fresh GSNO an increase was seen. That this increase was due to formation of GSNO, by reaction of some product of the decomposition with GSH, is made clear by Fig. 5. Thus admixture of decomposed GSNO with GSH increased both the 335 and the 544 nm absorbances that are characteristics of the spectrum of GSNO. Discussion The spontaneous decomposition of GSNO in the dark has been reported to yield oxidized glutathione (GSSG), glutathione disulfide S-oxide (GS(O)SG), glutathione disulfide S-dioxide (GS(O2)SG), glutathione sulfonic acid (GSO3H), and NO [15,16]. Although homolysis to GS plus NO could provide a conceptually simple mechanism, the thiyl was excluded as an
U
U
Fig. 2. Effect of dimedone on the decomposition of GSNO. 2.5 mM GSNO and 5 mM dimedone were incubated in PBS (pH 7.4) for 0–240 h at room temperature in the dark. UV/Vis spectra between 200 and 800 nm are shown in (A). These absorbances at 335 and 544 nm are plotted for 0–240 h in (B).
tive as the photolysis–chemiluminescence method. In order to repeat these measurements under the same conditions of Johnson et al. [8], the latter method was used and measurements were done by Dr. Hausladen in the Dr. Stamler laboratory [13]. Wild-type SOD1 and the FALS-associated mutant forms presented in Fig. 4 did not differ in their effects on the decomposition of 7 μM GSNO in the presence of 125 μM GSH in PBS (data not shown). We were thus unable to verify the result of Johnson et al. [8]. Autocatalysis of GSNO decomposition The great sensitivity of the rate of decomposition of GSNO to the initial concentration of GSNO, as well as the autocatalytic aspect of this rate of decomposition (Fig. 1), suggested that some product was able to increase the rate. The effect of decomposed GSNO on the rate of decomposition of fresh GSNO was therefore explored. Thus 5.0 mM GSNO was allowed to decompose in glass vessels in the dark for 11 days, at which time the residual absorbance at 335 nm was consistent with only 0.14 mM residual GSNO. This was mixed in various
Fig. 3. (A) Decomposition of 200 μM GSNO in the presence or absence of human wild-type SOD1 and increasing concentrations of GSH. 200 μM GSNO and 10 μM SOD1 were incubated in PBS (pH 7.4) for 30 min at room temperature in the dark in the presence of increasing concentrations of GSH. Each point represents the mean ± SD of triplicate samples. GSNO concentration was estimated from the molar extinction coefficient at 335 nm. (B) Initial rates of 200 μM GSNO decomposition as a function of SOD1 concentration in the presence of 2 mM GSH. Each point represents the mean ± SD of three to five samples.
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Fig. 4. No difference between effects of wild-type, FALS mutant, holo-, and apo-SOD1 on the decomposition of GSNO and the effect of polypropylene. 200 μM GSNO, 2 mM GSH, and 10 μM human SOD1 were mixed in PBS (pH 7.4) and 335 nm absorbance was measured for 15 min. The GSNO decomposition rate was calculated from the slopes. The concentration of 10 μM SOD1 was calculated from the molar extinction coefficient at 265 nm. The copper content of each SOD1 was estimated from the molar extinction coefficients at 265 and 655 nm and is given per subunit of SOD1 (gray bars). SOD activities are also shown (black bars).
intermediate by the failure of DMPO to trap it or to decrease the yield of GSSG [17]. Because trace metals have been seen to catalyze the decomposition of GSNO in the presence of U GSH, the displacement of NO from GSNO by Cu(I), or other metal cations present as impurities, may provide a plausible mechanism. Thus:
U
GSNO þ CuðIÞY GS−CuðIÞ þ NO ;
ð3Þ
GS−CuðIÞþ O2 þHþ Y GSO2 H þ CuðIIÞ;
ð4Þ
GSO2 H þ GSH X 2GSOH;
ð5Þ
GSOH þ GSNO YGSSG þHONO;
ð6Þ
HONO þ GSH YGSNO þ H2 O:
ð7Þ
This scheme allows for the formation of GSSG without U the involvement of GS . It involves the sulfenic acid as an intermediate, which could explain the reported inhibitory effect of dimedone [15]. NO as well as nitrous acid/nitrite accumulate and their reaction with GSH (Reaction (7)) could explain the formation of GSNO seen when decomposed GSNO was mixed with GSH. The reaction of the sulfenic acid with GSNO (Reaction (6)) could explain the observed autocatalysis. What can we conclude about the putative denitrosylase activity of SOD1? The very weakness of this activity plus its dependence on GSH and its inhibition by chelating agents, which have no effect on its dismutase activity, suggests that it is
due to extraneous metals bound at sites other than the active site. Recently, Ye and English reported that EDTA and DTPA bind to the active-site copper of one subunit without removing the metal, whereas they did not bind to apo-SOD1 [18]. They speculate this might induce a conformational change at the second active site that inhibits the denitrosylase activity but not the superoxide dismutase activity of the enzyme. However, apo-, H46R, and H48Q-SOD1 also had the denitrosylase activities (Fig. 4) that were inhibited by EDTA. Of course, they did not have superoxide dismutase activities (Fig. 4). It is likely that dialysis of SOD1 against EDTA or DTPA would eliminate bound extraneous metals, but subsequent placement of this enzyme in phosphate buffers would allow regain of the extraneous metals. Our prepared apo-SOD1 has trace Cu which shows weak superoxide dismutase activity (Fig. 4), which might allow it to have a denitrosylase activity. In any case, the claim that the FALS-associated SOD1s exhibit greater denitrosylase activity than the wild-type enzyme is not supported by our results. It must also be added that the in vivo environment is replete with chelating agents and because they inhibit the putative denitrosylase activity, it can have no in vivo relevance. Furthermore, we and others [7,8] showed that the maximal effect of GSH on the denitrosylase activity occurred when GSH concentration exceeded GSNO concentration by a factor of 10 (Fig. 3), but the maximum rate of decomposition of GSNO was still very slow. The putative denitrosylase activity of SOD1 was given in vivo relevance by the report that protein and peptide S-nitrosylation was decreased in FALS model mice [19]. It is unclear whether the reported decreased nitrosylation was due to the SOD1 or to something else. Moreover the consumption of GSNO by
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Fig. 5. Effects of decomposed GSNO. GSNO initially at 5.0 mM in PBS was allowed to decompose in the dark for 11 days. The absorbance at 335 nm then indicated that 0.14 mM GSNO remained. This 97% decomposed GSNO was then mixed, in various proportions, with fresh GSNO to adjust total GSNO concentration at 0.14 mM, in the presence of 1.4 mM GSH in PBS. The rates of change of absorbance at 335 nm are presented in (A). (B) shows the UV/Vis spectra of decomposed GSNO (D-GSNO), fresh GSNO (F-GSNO), and decomposed GSNO plus 1.4 mM GSH (D-GSNO + GSH).
SOD1, even if real, would be miniscule compared to that caused by GSNO reductase [20]. The ability of polypropylene to catalyze the decomposition of GSNO has been reported by Buettner et al. [21]. This is evidently due to some additive that can be washed from the surface of the polypropylene [21]. It is obvious that plastic vessels should not be used when manipulating GSNO. We also had the problem that disposable plastic pipette tips catalyze the decomposition of GSNO. Bulk tips have a risk of contamination from gloves and/or the reused tip case. Acknowledgments This work was supported by National Institutes of Health Research Grants R21-ES013682. We thank Drs. Alfred Hausladen, Akio Matsumoto, and Jonathan S. Stamler (Duke University Medical Center, Durham, NC, USA) for their technical assistance and helpful discussion. We are grateful to Dr. E. R. Stadtman (National Institutes of Health, Bethesda, MD, USA) for providing us the pVL1393-WThSOD1 and pVL1393-G93AhSOD1.
References [1] McCord, J. M.; Fridovich, I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244:6049–6055; 1969. [2] Beyer, W. F., Jr.; Fridovich, I. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal. Biochem. 161:559–566; 1987. [3] Fridovich, I.; Handler, P. Xanthine oxidase: V. Differential inhibition of the reduction of various electron acceptors. J. Biol. Chem. 237:916–921; 1962. [4] Hodgson, E. K.; Fridovich, I. The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: chemiluminescence and peroxidation. Biochemistry 14:5299–5303; 1975. [5] Hodgson, E. K.; Fridovich, I. The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme. Biochemistry 14:5294–5299; 1975. [6] Liochev, S. I.; Fridovich, I. CO2, not HCO−3 , facilitates oxidations by Cu, Zn superoxide dismutase plus H2O2. Proc. Natl. Acad. Sci. USA 101: 743–744; 2004. [7] Jourd'heuil, D.; Laroux, F. S.; Miles, A. M.; Wink, D. A.; Grisham, M. B. Effect of superoxide dismutase on the stability of S-nitrosothiols. Arch. Biochem. Biophys. 361:323–330; 1999. [8] Johnson, M. A.; Macdonald, T. L.; Mannick, J. B.; Conaway, M. R.; Gaston, B. Accelerated S-nitrosothiol breakdown by amyotrophic lateral sclerosis mutant copper,zinc-superoxide dismutase. J. Biol. Chem. 276:39872–39878; 2001.
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[9] Stamler, J. S.; Feelisch, M. Preparation and detection of S-nitrosothiols. Chichester, UK: Wiley; 1996. [10] Liochev, S. I.; Chen, L. L.; Hallewell, R. A.; Fridovich, I. Superoxidedependent peroxidase activity of H48Q: a superoxide dismutase variant associated with familial amyotrophic lateral sclerosis. Arch. Biochem. Biophys. 346:263–268; 1997. [11] Yim, M. B.; Kang, J. H.; Yim, H. S.; Kwak, H. S.; Chock, P. B.; Stadtman, E. R. A gain-of-function of an amyotrophic lateral sclerosis-associated Cu, Zn-superoxide dismutase mutant: an enhancement of free radical formation due to a decrease in Km for hydrogen peroxide. Proc. Natl. Acad. Sci. USA 93:5709–5714; 1996. [12] Stansell, M. J.; Deutsch, H. F. Physicochemical studies of crystalline human erythrocuprein. J. Biol. Chem. 240:4306–4311; 1965. [13] Mannick, J. B.; Hausladen, A.; Liu, L.; Hess, D. T.; Zeng, M.; Miao, Q. X.; Kane, L. S.; Gow, A. J.; Stamler, J. S. Fas-induced caspase denitrosylation. Science 284:651–654; 1999. [14] Frisell, W. R.; Mackenzie, C. G. The determination of formaldehyde and serine in biological systems. Methods Biochem. Anal. 6:63–77; 1958. [15] Tao, L.; English, A. M. Protein S-glutathiolation triggered by decomposed S-nitrosoglutathione. Biochemistry 43:4028–4038; 2004.
[16] Li, J.; Huang, F. L.; Huang, K. P. Glutathiolation of proteins by glutathione disulfide S-oxide derived from S-nitrosoglutathione: modifications of rat brain neurogranin/RC3 and neuromodulin/GAP-43. J. Biol. Chem. 276:3098–3105; 2001. [17] Singh, R. J.; Hogg, N.; Joseph, J.; Kalyanaraman, B. Mechanism of nitric oxide release from S-nitrosothiols. J. Biol. Chem. 271:18596–18603; 1996. [18] Ye, M.; English, A. M. Binding of polyaminocarboxylate chelators to the active-site copper inhibits the GSNO-reductase activity but not the superoxide dismutase activity of Cu,Zn-superoxide dismutase. Biochemistry 45:12723–12732; 2006. [19] Schonhoff, C. M.; Matsuoka, M.; Tummala, H.; Johnson, M. A.; Estevez, A. G.; Wu, R.; Kamaid, A.; Ricart, K. C.; Hashimoto, Y.; Gaston, B.; Macdonald, T. L.; Xu, Z.; Mannick, J. B. S-nitrosothiol depletion in amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. USA 103:2404–2409; 2006. [20] Liu, L.; Hausladen, A.; Zeng, M.; Que, L.; Heitman, J.; Stamler, J. S. A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans. Nature 410:490–494; 2001. [21] Buettner, G. R.; Scott, B. D.; Kerber, R. E.; Mugge, A. Free radicals from plastic syringes. Free Radic. Biol. Med. 11:69–70; 1991.
Original Contribution
Putative denitrosylase activity of Cu,Zn-superoxide dismutase Ayako Okado-Matsumoto 1 , Irwin Fridovich ⁎ Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA Received 20 April 2007; revised 23 May 2007; accepted 30 May 2007 Available online 15 June 2007
Abstract The Cu,Zn-superoxide dismutase (SOD1) has been reported to exert an S-nitrosylated glutathione (GSNO) denitrosylase activity that was augmented by a familial amyotrophic lateral sclerosis (FALS)-associated mutation in this enzyme. This putative enzymatic activity as well as the spontaneous decomposition of GSNO has been reexamined. The spontaneous decomposition of GSNO exhibited several peculiarities, such as a lag phase followed by an accelerating rate plus a marked dependence on GSNO concentration, suggestive of autocatalysis, and a greater rate in polypropylene than in glass vessels. Dimedone caused a rapid increase in absorbance likely due to reaction with GSNO, followed by a slower increase possibly due to reaction with an intermediate such as glutathione sulfenic acid. SOD1 weakly increased the rate of decomposition of GSNO, but did so only when GSH was present; and FALS-associated mutant forms of SOD1 were not more active in this regard than was the wild type. Decomposed GSNO, when added to fresh GSNO, hastened its decomposition, in accord with autocatalysis, and when added to GSH, generated GSNO in accord with the presence of nitrite. A mechanism is proposed that is in accord with these observations. © 2007 Elsevier Inc. All rights reserved. Keywords: Cu,Zn-superoxide dismutase; Amyotrophic lateral sclerosis; S-nitrosylated glutathione; Denitrosylase activity; Free radicals
The Cu,Zn-superoxide dismutase (SOD1) converts superU oxide (O2 − ) into oxygen plus hydrogen peroxide (H2O2) utilizing a catalytic cycle in which the active site Cu(II) is alternately reduced and reoxidized during successive encounters U with O2 − as in Reactions (1) and (2):
U
Enzyme−CuðIIÞþ O2 YEnzyme−CuðIÞþ O2
ð1Þ
Enzyme−CuðIÞ þ O2 þ Hþ Y Enzyme−CuðIIÞþ HO 2
ð2Þ
U
The HO2− product of Reaction (2) then protonates to H2O2. The assay, as originally defined [1], utilized 10 μM ferricyto-
U
Abbreviations: SOD1, Cu,Zn-superoxide dismutase; FALS, familial amyotrophic lateral sclerosis; O2 −, superoxide; GSH, glutathione; GSNO, S-nitrosylated glutathione; GSSG, oxidized glutathione; dimedone, 5,5dimethyl-1,3-cyclohexanedione; EDTA, ethylenediaminetetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; PBS, phosphate-buffered saline; DMPO, 5,5-dimethyl-1-pyrroline N-oxide. ⁎ Corresponding author. Fax: +1 919 684 8885. E-mail address: [email protected] (I. Fridovich). 1 Current address: Department of Biochemistry, Osaka University Graduate School of Medicine, Osaka 565-0872, Japan. 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.05.038
chrome c, 50 μM xanthine, and 100 μM EDTA in 3.0 ml of 50 mM potassium phosphate at pH 7.8 at 25°C. Sufficient xanthine oxidase was added to cause ΔA550 nm of 0.025 per minute, due to the reduction of the cytochrome c by the flux of U O2 − produced by the xanthine oxidase reaction. Samples containing SOD inhibited the ΔA550 nm and 1 unit was defined as that amount which halved that ΔA550 nm. Possible interferences with this assay and means of detecting and avoiding them have been described [2]. Xanthine oxidase, during catalytic turnover, is subject to inactivation by metal contaminants in the buffer, and the EDTA prevents that inactivation [3], without interfering with the activity of SOD. In addition to its activity as a superoxide dismutase, SOD1 has been seen to exert a nonspecific peroxidase activity [4,5] that is CO2-dependent at neutral pH [6]. Another activity, as a weak GSH-dependent denitrosylase, has been reported [7]. This putative activity gained importance from the report that familial amyotrophic lateral sclerosis (FALS)-associated mutant forms of SOD1 exerted this activity to a greater degree than did the wildtype enzyme [8]. In both of these reports [7,8], chelating agents, such as EDTA, were reported to inhibit the denitrosylase activity. Because EDTA does not interfere with the dismutation activity
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of SOD1, it seems that it cannot access the active-site copper. Hence its ability to inhibit the denitrosylase activity suggests that this activity may not involve this active-site copper. This reasoning led us to examine the denitrosylase reactivity of SOD1. Herein we report that this activity is trivially weak and moreover note that there is no difference in this activity between the FALS-associated mutant and the wild-type forms of SOD1. Furthermore, apo-SOD1, H46R and H48Q that have a Cubinding site mutation, also had such denitrosylase activity, even though they did not have superoxide dismutase activity. We also note that decomposition of S-nitrosylated glutathione (GSNO) was more rapid in polypropylene than in glass vessels and that partially decomposed GSNO accelerated the decomposition of fresh GSNO and that largely decomposed GSNO reacts with GSH to regenerate GSNO. A mechanism is proposed that is in accord with these observations.
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metrically and by photolysis chemiluminescence. The decomposition of 200 μM GSNO with 2 mM GSH in the presence and absence of 10 μM SOD1 in PBS was measured as a function of time. The decomposition of 7 μM GSNO with 125 μM GSH in the presence and absence of 10 μM SOD1 in PBS was followed by photolysis chemiluminescence using a Nitrolite photolysis unit (Thermo Orion) and TEA 510 analyzer (Thermo Orion) [13]. Every 4 min for 24 min, 25 μl of reaction mixture was injected in the Nitrolite and total photolyzable NO species were measured. Superoxide dismutase activity Superoxide dismutase activities of SOD1 were measured by cytochrome c assay with 100 μM EDTA [1]. Results
Materials and methods Decomposition of GSNO at pH 7.4 Preparation of GSNO Fresh GSNO was synthesized as described by Jourd'heuil [7] with minor modification. Equal volumes of 1 M GSH in 1 N HCl and 1 M sodium nitrite in water were mixed for 30 min at room temperature in the dark. Then the solutions were neutralized with 10 N NaOH and diluted a minimum of 25-fold into phosphatebuffered saline (PBS) at pH 7.4 (Gibco) and kept on ice in dark. Decomposed GSNO was prepared by storing fresh 5 mM GSNO solution at room temperature in a glass vessel in the dark for 11 days. GSNO concentration was estimated using the molar extinction coefficients at 335 nm (586 M−1 cm−1) or 544 nm (17.2 M−1 cm−1) [9]. Preparation of wild-type and FALS-associated mutant human SOD1 Free cysteine-mutated (C6A and C111S) wild-type (WT), G93A, G93R, and E100G human SOD1 was produced and purified as described previously [10]. We expressed WT and G93A human SOD1 using a baculovirus vector in Spodoptera frugiperda (Sf9) cells. pVL1393-WTSOD1 and pVL1393G93ASOD1 were provided by Dr. E. R. Stadtman [11] and were cotransfected with linearized baculoviral DNA (PharMingen) into Sf9 cells. We then purified the SOD1 with DE52, CM52 (Whatman), and gel filtration (Ultrogel AcA54; Sigma). The concentrations of hSOD1 were estimated using the molar extinction coefficient at 265 nm (18,700 M−1 cm−1) [12], and copper content per subunit was estimated from the difference between the molar extinction coefficients at 265 and 655 nm (350 M−1 cm−1) [12].
In PBS at pH 7.4 at room temperature, GSNO exhibits a strong absorbance centered at 335 nm and a weaker band centered at 544 nm. The decomposition of GSNO could be conveniently followed spectrophotometrically and was faster when the mixture was in contact with polypropylene compared to in glass vessels. GSNO initially at 5.0 mM decomposed strikingly faster than did a solution initially at 2.5 mM. Moreover, the decomposition of the 5.0 mM solutions exhibited an initial lag followed by an accelerating rate. These characteristics are shown in Figs. 1A–1D and suggest a mechanism in which a product or an accumulated intermediate can augment the rate of consumption of GSNO. Effect of dimedone Dimedone, long used for the detection of aldehydes [14], apparently also reacts with sulfenic acids [15]. Dimedone at 5.0 mM absorbs strongly below 320 nm and, when mixed with 2.5 mM GSNO, caused a rapid increase in absorbance at 325 and 544 nm in PBS (pH 7.4), followed by a much slower increase; as shown in Figs. 2A and 2B. Tao and English reported that GSNO decomposition in the dark slowed dramatically when dimedone was present in GSNO solution in water, and an ∼10% loss of GSNO absorbance at 550 nm was observed after 51 h [15]. We reexamined the effect of dimedone in PBS (pH 7.4) instead of water because the stability of GSNO might be pH-dependent. It seems likely that the rapid increase in absorbance was due to the reaction of dimedone with GSNO in PBS (pH 7.4), whereas the slower increase was due to the reaction of dimedone with some product, or intermediate, of this GSNO decomposition.
Decomposition of GSNO Effects of GSH and SOD1 GSNO at 2.5 and 5 mM in PBS (pH 7.4) was stored at room temperature in the dark and UV/Vis spectra were measured using a UV-2501 or UV-2550 Shimadzu spectrophotometer. The denitrosylase activities of SOD1 were measured spectrophoto-
GSH added to GSNO had an insignificant effect on its rate of decomposition. However, when added in the presence of 10 μM SOD1, it increased that rate, but only when GSH was
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Fig. 1. Spontaneous decomposition of GSNO. UV/Vis spectra between 200 and 800 nm of (A) 2.5 mM and (B) 5 mM GSNO in PBS (pH 7.4) after standing at room temperature in the dark for 0–240 h (0, 72, 120, 144, and 240 are shown) in glass (G) and in polypropylene plastic (P) tubes are shown. The inset shows the absorbance between 500 and 600 nm. GSNO exhibits an absorption band centered at ∼ 335 nm and a weaker ∼ 544 nm band. These absorbances at 335 and 544 nm of (C) 2.5 mM and (D) 5 mM GSNO are plotted for 0–240 h.
in excess of GSNO. This is seen in Fig. 3A. The maximal effect of GSH occurred when GSH concentration exceeded GSNO concentration by a factor of 10, but even then the rate of decomposition of GSNO was very slow. It may well be that the requirement for excess GSH is explained by the reactive form being the thiolate anion rather than the thiol. Because the pKa of the thiol in GSH is 9.12, only a bit more than 1% would be ionized at pH 7.4. The same phenomenon was reported by Jourd'heuil et al. [7] and Johnson et al. [8]. We also found that the initial rate of GSNO decomposition in the presence of 2 mM GSH, 200 μM GSNO, and increasing concentration from 3 to 30 μM SOD1 followed saturation kinetics; however, less than 1 μM SOD1 did not significantly affect GSNO decomposition (Fig. 3B). Effect of SOD1 SOD1 has been reported to catalyze the denitrosylation of GSNO [7], and FALS-related SOD1 mutants have been stated to
exhibit more of this activity [8]. Actually, because the SOD1 has been applied at 10 μM or more, true catalytic turnover has yet to be seen. Fig. 4 presents the effects of several forms of SOD1 at 10 μM on the rate of decomposition of 200 μM GSNO in the presence of 2 mM GSH. Several things are immediately apparent. These are: (a) the rate of decomposition of GSNO is not more than doubled by these enzymes; (b) the mutant forms were not more active than the wild type; (c) the weak effect of SOD1 seen in glass vessels in not seen when the reaction occurred in polypropylene vessels; (d) replacing the two sulfhydryl-bearing cysteine residues of human SOD1 by alanine (C6A) and serine (C111S), which precludes glutathionylation and/or transnitrosation, was without effect; and (e) the copper contents, estimated from the molar extinction coefficients determined by Stansell and Deutsch [12], did not correlate with the apparent denitrosylase activity. It follows that the toxic gain of function of the FALS-related mutant forms of SOD1 cannot be a gain of denitrosylase activity, as has been claimed [8]. The spectrophotometric detection of GSNO is not as sensi-
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proportions with 0.14 mM fresh GSNO, in the presence of 1.4 mM GSH, and rates of change of absorbance at 335 nm were followed. Fig. 5 shows that an admixture of small amounts of aged GSNO with fresh GSNO plus GSH caused a decline in absorbance at 335 nm, whereas at high ratios of the aged to the fresh GSNO an increase was seen. That this increase was due to formation of GSNO, by reaction of some product of the decomposition with GSH, is made clear by Fig. 5. Thus admixture of decomposed GSNO with GSH increased both the 335 and the 544 nm absorbances that are characteristics of the spectrum of GSNO. Discussion The spontaneous decomposition of GSNO in the dark has been reported to yield oxidized glutathione (GSSG), glutathione disulfide S-oxide (GS(O)SG), glutathione disulfide S-dioxide (GS(O2)SG), glutathione sulfonic acid (GSO3H), and NO [15,16]. Although homolysis to GS plus NO could provide a conceptually simple mechanism, the thiyl was excluded as an
U
U
Fig. 2. Effect of dimedone on the decomposition of GSNO. 2.5 mM GSNO and 5 mM dimedone were incubated in PBS (pH 7.4) for 0–240 h at room temperature in the dark. UV/Vis spectra between 200 and 800 nm are shown in (A). These absorbances at 335 and 544 nm are plotted for 0–240 h in (B).
tive as the photolysis–chemiluminescence method. In order to repeat these measurements under the same conditions of Johnson et al. [8], the latter method was used and measurements were done by Dr. Hausladen in the Dr. Stamler laboratory [13]. Wild-type SOD1 and the FALS-associated mutant forms presented in Fig. 4 did not differ in their effects on the decomposition of 7 μM GSNO in the presence of 125 μM GSH in PBS (data not shown). We were thus unable to verify the result of Johnson et al. [8]. Autocatalysis of GSNO decomposition The great sensitivity of the rate of decomposition of GSNO to the initial concentration of GSNO, as well as the autocatalytic aspect of this rate of decomposition (Fig. 1), suggested that some product was able to increase the rate. The effect of decomposed GSNO on the rate of decomposition of fresh GSNO was therefore explored. Thus 5.0 mM GSNO was allowed to decompose in glass vessels in the dark for 11 days, at which time the residual absorbance at 335 nm was consistent with only 0.14 mM residual GSNO. This was mixed in various
Fig. 3. (A) Decomposition of 200 μM GSNO in the presence or absence of human wild-type SOD1 and increasing concentrations of GSH. 200 μM GSNO and 10 μM SOD1 were incubated in PBS (pH 7.4) for 30 min at room temperature in the dark in the presence of increasing concentrations of GSH. Each point represents the mean ± SD of triplicate samples. GSNO concentration was estimated from the molar extinction coefficient at 335 nm. (B) Initial rates of 200 μM GSNO decomposition as a function of SOD1 concentration in the presence of 2 mM GSH. Each point represents the mean ± SD of three to five samples.
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Fig. 4. No difference between effects of wild-type, FALS mutant, holo-, and apo-SOD1 on the decomposition of GSNO and the effect of polypropylene. 200 μM GSNO, 2 mM GSH, and 10 μM human SOD1 were mixed in PBS (pH 7.4) and 335 nm absorbance was measured for 15 min. The GSNO decomposition rate was calculated from the slopes. The concentration of 10 μM SOD1 was calculated from the molar extinction coefficient at 265 nm. The copper content of each SOD1 was estimated from the molar extinction coefficients at 265 and 655 nm and is given per subunit of SOD1 (gray bars). SOD activities are also shown (black bars).
intermediate by the failure of DMPO to trap it or to decrease the yield of GSSG [17]. Because trace metals have been seen to catalyze the decomposition of GSNO in the presence of U GSH, the displacement of NO from GSNO by Cu(I), or other metal cations present as impurities, may provide a plausible mechanism. Thus:
U
GSNO þ CuðIÞY GS−CuðIÞ þ NO ;
ð3Þ
GS−CuðIÞþ O2 þHþ Y GSO2 H þ CuðIIÞ;
ð4Þ
GSO2 H þ GSH X 2GSOH;
ð5Þ
GSOH þ GSNO YGSSG þHONO;
ð6Þ
HONO þ GSH YGSNO þ H2 O:
ð7Þ
This scheme allows for the formation of GSSG without U the involvement of GS . It involves the sulfenic acid as an intermediate, which could explain the reported inhibitory effect of dimedone [15]. NO as well as nitrous acid/nitrite accumulate and their reaction with GSH (Reaction (7)) could explain the formation of GSNO seen when decomposed GSNO was mixed with GSH. The reaction of the sulfenic acid with GSNO (Reaction (6)) could explain the observed autocatalysis. What can we conclude about the putative denitrosylase activity of SOD1? The very weakness of this activity plus its dependence on GSH and its inhibition by chelating agents, which have no effect on its dismutase activity, suggests that it is
due to extraneous metals bound at sites other than the active site. Recently, Ye and English reported that EDTA and DTPA bind to the active-site copper of one subunit without removing the metal, whereas they did not bind to apo-SOD1 [18]. They speculate this might induce a conformational change at the second active site that inhibits the denitrosylase activity but not the superoxide dismutase activity of the enzyme. However, apo-, H46R, and H48Q-SOD1 also had the denitrosylase activities (Fig. 4) that were inhibited by EDTA. Of course, they did not have superoxide dismutase activities (Fig. 4). It is likely that dialysis of SOD1 against EDTA or DTPA would eliminate bound extraneous metals, but subsequent placement of this enzyme in phosphate buffers would allow regain of the extraneous metals. Our prepared apo-SOD1 has trace Cu which shows weak superoxide dismutase activity (Fig. 4), which might allow it to have a denitrosylase activity. In any case, the claim that the FALS-associated SOD1s exhibit greater denitrosylase activity than the wild-type enzyme is not supported by our results. It must also be added that the in vivo environment is replete with chelating agents and because they inhibit the putative denitrosylase activity, it can have no in vivo relevance. Furthermore, we and others [7,8] showed that the maximal effect of GSH on the denitrosylase activity occurred when GSH concentration exceeded GSNO concentration by a factor of 10 (Fig. 3), but the maximum rate of decomposition of GSNO was still very slow. The putative denitrosylase activity of SOD1 was given in vivo relevance by the report that protein and peptide S-nitrosylation was decreased in FALS model mice [19]. It is unclear whether the reported decreased nitrosylation was due to the SOD1 or to something else. Moreover the consumption of GSNO by
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Fig. 5. Effects of decomposed GSNO. GSNO initially at 5.0 mM in PBS was allowed to decompose in the dark for 11 days. The absorbance at 335 nm then indicated that 0.14 mM GSNO remained. This 97% decomposed GSNO was then mixed, in various proportions, with fresh GSNO to adjust total GSNO concentration at 0.14 mM, in the presence of 1.4 mM GSH in PBS. The rates of change of absorbance at 335 nm are presented in (A). (B) shows the UV/Vis spectra of decomposed GSNO (D-GSNO), fresh GSNO (F-GSNO), and decomposed GSNO plus 1.4 mM GSH (D-GSNO + GSH).
SOD1, even if real, would be miniscule compared to that caused by GSNO reductase [20]. The ability of polypropylene to catalyze the decomposition of GSNO has been reported by Buettner et al. [21]. This is evidently due to some additive that can be washed from the surface of the polypropylene [21]. It is obvious that plastic vessels should not be used when manipulating GSNO. We also had the problem that disposable plastic pipette tips catalyze the decomposition of GSNO. Bulk tips have a risk of contamination from gloves and/or the reused tip case. Acknowledgments This work was supported by National Institutes of Health Research Grants R21-ES013682. We thank Drs. Alfred Hausladen, Akio Matsumoto, and Jonathan S. Stamler (Duke University Medical Center, Durham, NC, USA) for their technical assistance and helpful discussion. We are grateful to Dr. E. R. Stadtman (National Institutes of Health, Bethesda, MD, USA) for providing us the pVL1393-WThSOD1 and pVL1393-G93AhSOD1.
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