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Mechanisms of Nitrogen Oxide-Mediated Disruption of Metalloprotein Function: An Examination of the Copper-Responsive Yeast Transcription Factor Ace1 Masaru Shinyashiki,† Chuan-Ju G. Pan,† Christopher H. Switzer,‡ and Jon M. Fukuto*,† Department of Pharmacology, UCLA School of Medicine, Center for the Health Sciences, Los Angeles, California 90095-1735, and Department of Chemistry and Biochemistry, University of CaliforniasLos Angeles, Los Angeles, California 90095-1569 Received June 13, 2001
Nitric oxide (NO) has been found to inhibit the copper-responsive yeast transcription factor Ace1 in an oxygen-dependent manner. However, the mechanism responsible for NO-dependent inhibition of Ace1 remains unestablished. In the present study, the chemical interaction of nitrogen oxide species with Ace1 was examined using a yeast reporter system. Exposure of yeast to various nitrogen oxides, under a variety of conditions, revealed that the oxygendependent inhibition of Ace1 is due to the reaction of NO with O2. The nitrosating nitrogen oxide species N2O3 is likely to be the disrupter of Ace1 activity. Considering the similarity of metal-thiolate ligation in Ace1 with other mammalian metalloproteins such as metallothionein, metal chaperones, and zinc-finger proteins, these results help to understand the biochemical interactions of NO with those mammalian metalloproteins.
Introduction (NO)1
Nitric oxide and related nitrogen oxide species are generated endogenously under a variety of physiological circumstances. Nitric oxide can be a physiological messenger via activation of soluble guanylate cyclase, leading to smooth muscle relaxation (for example, see refs 1 and 2) or it can be a vital part of the immune system response to a variety of pathogens (for example, see ref 3). As an immunological agent, NO is presumed to be either cytostatic or cytotoxic by, as yet, unestablished mechanisms. It has been reported that NO is capable of disrupting metal ion-homeostasis (or metal metabolism) and this may represent a mechanism by which NO can disrupt or regulate cell function. For example, disruption of iron metabolism by NO can occur through its interaction with the iron-sulfur cluster of iron regulatory proteins (see ref 4 and references therein) leading to changes in ferritin or transferrin mRNA stability. Recently, it has been proposed that NO and/or NO-derived species can disrupt/regulate gene expression by interacting with DNA-binding metalloproteins. For example, zinc-finger proteins are sites of possible nitrogen oxide interactions (5). Our lab has recently reported that the copper-binding and copper-responsive yeast Saccharomyces cerevisiae transcription factor Ace1 can be inhibited by exposure to NO (6), which leads to an increase in * To whom correspondence should be addressed. Fax: (310) 8256267. E-mail:
[email protected]. † Department of Pharmacology. ‡ Department of Chemistry and Biochemistry. 1 Abbreviations: NO, nitric oxide; -OONO, peroxynitrite; O -, 2 superoxide; ONPG, o-nitrophenyl β-D-galactopyranoside; BCS, bathocuproinedisulfonic acid; BSA, bovine serum albumin; PTIO, 2-phenyl4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; SD - ura, synthetic dextrose medium minus Ura; DDW, double distilled water; DMSO, dimethyl sulfoxide.
yeast susceptibility to copper toxicity (presumably by inhibiting the expression of protective metal metabolism proteins) (7). The yeast transcription factor Ace1 is representative of a class of copper-binding proteins which bind copper via thiolate ligation. For example, a variety of human and yeast proteins which serve to chaperone, transport, and sequester metals have been discovered which bind metals in a similar or homologous manner to that found in Ace1 (8-11). Thus, elucidation of the chemical interactions of nitrogen oxides with Ace1 will serve as a basis for understanding possible interactions with analogous proteins and, possibly, provide a chemical rationale for NO-mediated toxicity or gene disruption. The utility of the yeast S. cerevisiae system for examining the details of biological NO chemistry stems from the fact that they can grow/survive under a variety of conditions. Thus, exposure of yeast to nitrogen oxides can be carried out under diverse conditions (at least compared to similar studies in mammalian cell systems) which either preclude or favor certain types of nitrogen oxide chemistry. For example, yeast will flourish under either aerobic or anaerobic conditions allowing the O2-dependency of NO-mediated effects to be evaluated. Moreover, yeast can also grow under a widely varying pH range, allowing the pH-dependence of NO-mediated events to be examined. Although yeast possess these important differences compared to mammalian cells, they maintain important similarities as well, making them an ideal model system for mammalian systems (vida infra). Herein, we describe a series of studies in yeast aimed at elucidating the nature of the chemical interactions of nitric oxide, and related nitrogen oxides, with a representative copper-binding metalloprotein, Ace1.
10.1021/tx010102i CCC: $20.00 © 2001 American Chemical Society Published on Web 11/28/2001
Mechanism of Ace1 Inhibition by NO
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Experimental Procedures Chemicals. Yeast nitrogen base, D-glucose, amino acids, adenine, uracil, o-nitrophenyl β-D-galactopyranoside (ONPG), bathocuproinedisulfonic acid (BCS), and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). 2-Phenyl4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) was purchased from Calbiochem (La Jolla, CA). Nitric oxide was purchased from Praxair (Burr Ridge, IL). Nitrogen and NO2 gas were purchased from Puritan-Bennett (Overland Park, KS). Protein Assay Dye Reagent Concentrate was purchased from Bio-Rad (Hercules, CA). All other chemicals and solutions were purchased from commercial suppliers and were of the highest purity available. Yeast Culture, CUP1-lacZ Induction, and β-Galactosidase Assay. To examine the effects of nitrogen oxide species on the copper-responsive transcription factor Ace1, we utilized a reporter plasmid based on CUP1 promotor and lacZ fusion (12). 217CUP was prepared by transforming wild-type strain EG217 (W303A, MATa leu2-3,112 trp1-1 his3-11,15 ura3-1 ade2-1) with CUP1 promoter/lacZ (β-galactosidase) fusion reporter plasmid as described (7). Cells were inoculated (start OD600 ) 0.01-0.05) in synthetic dextrose medium minus uracil (SD - ura) that had trace copper removed by the method of Chiang et al. (7) (SD - ura - Cu) and which contained 0.1 mM BCS. The cells were grown at 30 °C with shaking (275 rpm) to late log phase (OD600 ) 3-3.5) and then used for all experiments. To induce Ace1-promoted β-galactosidase, the cells were washed twice with double-distilled water (DDW) and resuspended in SD - ura + CuSO4 (0.1 mM), then incubated at 30 °C for 60 min with shaking (275 rpm). After induction, cells were washed with DDW and stored at -80 °C until use. Yeast cells were defrosted on ice and disrupted in Z buffer (100 mM sodium phosphate buffer, pH 7, 10 mM KCl, 1 mM MgSO4, and 50 mM 2-mercaptoethanol) with glass beads (0.5 mm diameter), and centrifuged at 15000g for 5 min at 4 °C. The supernatants were used for β-galactosidase assay. β-Galactosidase activity was determined using ONPG by the colorimetric assay previously described (7). Protein concentration in the cell extracts was measured by the method of Bradford (13) using BSA as a standard. β-Galactosidase activity is expressed as units defined as (Abs420nm)(1000)/time (min)/mg of protein. Anaerobic Experiments. The effects of nitrogen oxide species were tested under both aerobic and anaerobic conditions. To establish anaerobic conditions, 217CUP cells (10 mL, OD600 ) 3-3.5) were transferred to septum-sealed vials (details are described in each figure legend). To remove oxygen from the growth medium, nitrogen gas was continuously purged through the headspace via inlet and outlet needles through the septum for 30-45 min. as previously described (6). Experiments run under aerobic conditions were performed similarly except no N2 purge was carried out and the incubations were performed in the vials sealed under an atmosphere of air. For anaerobic experiments, all chemicals were injected using gastight syringes, and the cells were incubated at 30 °C with shaking. After exposure of the cells to the various agents was complete, the system was purged with nitrogen gas continuously for 15-30 min before opening the rubber septum to remove any residual NO. The cells were then washed and exposed to 0.1 mM of CuSO4 to induce β-galactosidase. For aerobic experiments, the vial caps were loosened before and/or during exposure to chemicals. Exposure of Yeast Cells to Nitrogen Oxide Species. Exposure of yeast to NO was accomplished using authentic NO gas, injected directly into the headspace of incubation mixtures. Prior to use, NO gas was passed through 1 N NaOH to remove contaminating species such as N2O3. Peroxynitrite (-OONO) was synthesized according to the method of Beckman et al. (14). The concentration of peroxynitrite was estimated using an extinction coefficient of 1670 M-1 cm-1 at 302 nm. Stock solutions of -OONO were prepared in 1.2 N NaOH. It was determined that addition of the stock -OONO solutions into the culture medium gave little change in the pH. Stock solutions of 10 and 100 mM
Figure 1. Oxygen-dependent inhibition of Ace1 by authentic NO gas. Late log phase culture of 217CUP (10 mL) was transferred into 35-mL scale glass centrifuge tubes sealed with silicon rubber septa. Aerobic and anaerobic conditions were made as described in Experimental Procedures. NO gas (0, 0.25, 0.5, and 1 mL) was injected anaerobically using gastight syringe into the headspace. After incubation for 10 min at 30 °C with shaking, N2 gas was purged through headspace for 15 min to remove remained NO gas. Induction of Ace1-promoted CUP1lacZ and β-galactosidase assay were performed as described in Experimental Procedures. Control (no NO gas) values of aerobic and anaerobic conditions are 16229 and 12553 units, respectively. Four independent experiments were performed and representative data are shown. PTIO (CalBiochem, La Jolla, CA) were prepared in dimethyl sulfoxide (DMSO). To generate NO2 under anaerobic conditions, NO gas was injected into the headspace of the yeast culture under anaerobic conditions followed by the addition of aliquot of PTIO to the 217CUP culture using a gastight syringe (final concentration of 0.1 and 1 mM). The cells were then incubated at 30 °C for 10 min with shaking. To examine the effects of N2O3, we utilized two different procedures to generate N2O3. Various amounts of NO and/or NO2 gas were injected into the incubation headspace of anaerobically prepared 217CUP cultures. The cell cultures were incubated at 30 °C for 10 min followed by degassing with N2. N2O3 was also generated in situ using acidified nitrite under aerobic conditions. Cells were washed with DDW twice and resuspended SD - ura + BCS (0.1 mM) at pH 4-7. NaNO2 (0-5 mM) was added into each suspension and the cells were incubated at 30 °C for 10 min. After incubations, the cells were assayed for β-galactosidase activity as described above. In all studies involving NO2 exposure the pH of solutions after exposure was determined. It was found that significant NO2 exposure will decrease the pH of incubation solutions (e.g., from pH 6 to pH 4 under the conditions of highest NO2 administration). However, control experiments indicate that these changes in pH do not affect the β-galactosidase activity or expression in our systems.
Results Oxygen-Dependent Inhibition of Ace1 by NO. We previously reported that NO, using a diazenium diolate NO-donor, inhibits Ace1 in a oxygen-dependent manner (6). Herein, the affect of authentic NO gas on Ace1 activity was examined. Thus, NO gas was directly injected into the headspace of sealed incubation flasks. Using this method of NO exposure, NO was found to inhibit Ace1 in a dose-dependent manner, and consistent with our previous observations, inhibition was O2-dependent (Figure 1). The oxygen-dependency for the inhibition of Ace1 by NO may be the result of a variety of possible mechanisms. For example, inhibition may be due to the generation of other nitrogen oxides generated from reaction of NO with O2 such as NO2 or N2O3 (reactions 1 and 2) or reaction of NO with the reduced oxygen species superoxide (O2-) to give the potent oxidant, -OONO (reaction 3) (15-17).
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Figure 2. Peroxynitrite is not capable of inhibiting Ace1 activity. Late log phase 217CUP cell cultures (10 mL) were exposed to peroxynitrite (0, 0.1, 0.2, 0.4, 0.6 and 0.8 mM) for 10 min at 30 °C with orbital shaking (275 rpm) under aerobic condition in 50-mL plastic centrifuge tubes. After induction of Ace1-promoted CUP1-lacZ, β-galactosidase activities were determined. Three independent experiments were performed and representative data are shown.
Alternatively, inhibition of Ace1 may be a result of two independent steps, one involving O2 and the other involving NO (discussed later).
2NO + O2 f 2NO2
(1)
NO2 + NO f N2O3
(2)
NO + O2- f -OONO
(3)
Inhibition of copper-dependent Ace1 activity by -OONO was examined. The effect of -OONO on Ace1 activity is shown in Figure 2. Exposure of 217CUP cells to -OONO at concentrations up to 0.8 mM prior to exposure to copper does not affect the copper-dependent activation of Ace1. Simultaneous exposure of yeast to copper and -OONO (at concentrations up to 0.8 mM) also exhibited no inhibition of Ace1 activation (data not shown). These data suggest that the reactions of NO with oxygen species other than O2- or the interaction of Ace1 with NO and O2 by two independent steps are responsible for the Ace1 inhibition. Other nitrogen oxide species were examined for their Ace1 inhibitory capacity. We tested the idea that NO2 generation is required for the NO-dependent Ace1 inhibition, by utilizing an in situ oxidant which is capable of converting NO to NO2 in an O2-independent fashion. The NO oxidant PTIO is known to stoichiometrically convert NO directly to NO2 (reaction 4) (18).
PTIO + NO f PTI + NO2
(4)
Thus, we examined the effects of PTIO on the NOdependent inhibition of copper-dependent Ace1 activation. Exposure of yeast to NO (via injection of NO gas into the incubation headspace) resulted in Ace1 inhibition under aerobic conditions (Figure 3). Simultaneous exposure of yeast to NO and PTIO (1 mM) had no effect on the observed aerobic inhibition of Ace1 by NO. However, under anaerobic conditions, NO was able to inhibit Ace1 in the presence of 1 mM PTIO. These results suggest that production of NO2 is required for the inhibition of copperdependent Ace1 activation by NO. The generation of NO2 appears to be required for copper-dependent Ace1 inhibition by NO. We then ex-
Figure 3. Inhibition of Ace1 by NO2 generated from oxygenindependent NO oxidation by PTIO. Aerobic and anaerobic conditions of late log phase 217CUP (10 mL) were prepared as described in Figure 1 legend. NO gas (1 mL/headspace) was injected into the headspace anaerobically. Then 100 µL of PTIO stock solution (10 or 100 mM in DMSO) was immediately injected into the culture. For the condition without PTIO, cultures were added with 100 µL of DMSO. The cultures were incubated at 30 °C for 10 min, then N2 was purged for 15 min. After induction of Ace1-promoted CUP1-lacZ, β-galactosidase activities were determined. Control (no NO gas) values for aerobic, aerobic plus PTIO (1 mM), anaerobic, anaerobic plus PTIO (0.1 mM), and anaerobic plus PTIO (1 mM) are 15839, 10954, 9939, 8943, and 6800 units, respectively. Three independent experiments were performed and representative data are shown.
amined the inhibitory capacity of other NO2-derived nitrogen oxides. As mentioned above, NO2 reacts with NO to give N2O3 (reaction 2) which is known to be a potent nitrosating agent (reaction 5).
N2O3 + Nuc-H f Nuc-NO + NO2- + H+
(5)
where Nuc ) nucleophile (i.e., thiols, amines, etc.). To examine the inhibitory activity of NO2 and other derivative nitrogen oxides such as N2O3, authentic NO and NO2 gas were injected into the headspace of anaerobic incubations of yeast. Thus, 217CUP cells were exposed to NO2 gas with or without the addition of NO gas. Exposure of yeast to NO2 gas alone exhibited some copper-dependent Ace1 inhibition under anaerobic conditions (Figure 4). Although NO alone did not inhibit Ace1 under anaerobic conditions, it did significantly enhance NO2-mediated inhibition when yeasts were exposed to NO and NO2 simultaneously. Another method for generating N2O3 in situ is via the acidification of NO2- (reaction 6 and 7).
NO2- + H+ f HONO
(6)
2HONO f N2O3 + H2O
(7)
Compared to mammalian cells, S. cerevisiae are capable of growing/surviving over a wide pH range and are, therefore, amenable to studies whereby the effects of pH on the activity of NO2- are examined. Thus, 217CUP cells were exposed to varying concentrations of NaNO2 (0-5 mM) at varying pH (4-7) for 10 min and then assayed for β-galactosidase activity. As shown in Figure 5, inhibition of copper-dependent Ace1 activity was observed at high concentrations of NaNO2 and at low pH.
Mechanism of Ace1 Inhibition by NO
Figure 4. Dose-dependent effect of NO2 and/or NO gas on Ace1 activity under anaerobic condition. Late log phase cultures of 217CUP (10 mL) were transferred into 25-mL Erlenmeyer flasks with rubber septa. Anaerobic conditions were made by the method described in Experimental Procedures. Various amounts of NO (0, 0.4, or 1 mL) and/or NO2 (0, 0.2, 0.4, or 1 mL) gases were injected into headspace of the cultures anaerobically. The cells were incubated at 30 °C for 10 min, N2 gas was then purged for 15 min. After induction of Ace1-promoted CUP1-lacZ, β-galactosidase activity was determined. Three independent experiments were performed and representative data are shown.
Figure 5. Inhibition of Ace1 by N2O3 generated from acidified NO2-. Late log phase 217CUP cells were washed with DDW twice and resuspended in different pH (4 and 7) of SD - ura + BCS (0.1 mM). Each culture (10 mL in plastic centrifuge tube) was added with various concentrations of NaNO2 (0, 0.2, 1, and 5 mM) and incubated at 30 °C for 10 min with orbital shaking (275 rpm) under aerobic condition. Then Ace1-promoted CUP1lacZ was induced and β-galactosidase activity was measured. Data are shown as mean ( of three independent experiments. Significant differences [(*) P < 0.01] were calculated by t-test.
Discussion We have previously shown that NO is capable of inhibiting the copper-dependent activity of the copperresponsive transcription factor Ace1 (6). Furthermore, we previously demonstrated that Ace1 inhibition by NO leads to a dramatic increase in the susceptibility of yeast to copper toxicity (7). This observed increase in copper toxicity is consistent with the demonstration by others that Ace1 activity is vital for protection of yeast from metal toxicity (19, 20). Thus, it is clear that NO (or an NO-derived species) is capable of altering the metalbinding capacity of Ace1 resulting in a loss of gene activating activity. However, the chemistry responsible for this inhibition is not established and is the subject of this study. The metal binding domain of Ace1 is similar to that found in the metal sequestering protein metallothionein (21, 22). Cuprous ion binds to Ace1 via ligation to multiple cysteine residues in the binding pocket. Presumably, metal ligation to Ace1 confers the proper
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conformation for DNA binding and gene activation. Our previous work implicates thiol ligand modification as a potential mechanism of NO-mediated disruption of copper-dependent Ace1 activity (6). Thus, we suspect that the ability of NO, or an NO-derived species, to modify the ligating thiols of Ace1 is responsible for the observed inhibition of activity. The interaction of NO, and related nitrogen oxides, with thiols/thiolates has been examined previously by others (23-27). However, the interaction of NO with metallothiol proteins (i.e., metal-bound thiols) has not been examined in rigorous detail nor has this chemistry been firmly established in a whole cell system. Consistent with our previous report (6), inhibition of copper-dependent Ace1 activity by NO is largely O2dependent (Figure 1). There are a number of mechanistic possibilities which can explain the observed O2-dependency. For example, intracellular O2 reduction may generate O2- which can react with NO (reaction 3) to form the potent thiol oxidizing agent, -OONO (24). Since copper binding to Ace1 occurs through thiolate ligation (10), it is not unreasonable to suspect that -OONOmediated oxidation of the thiolate ligands may be responsible for the observed NO-mediated inhibition of Ace activity. Moreover, previous work indicates that -OONO is capable of modifying the function of proteins via thiol modification (for example, see refs 28 and 29) and releasing thiolate-bound zinc from proteins (30). However, we believe that this is not the case in our studies since yeast treated with authentic -OONO did not exhibit any Ace1 inhibition at concentrations of -OONO up to 0.8 mM (Figure 2). Considering that 0.5 mM of the NOdonor DEA/NO completely inhibits copper-dependent Ace1 activation (6), it is unreasonable to think that 0.8 mM -OONO would not show some inhibition. Significantly, exogenous -OONO is capable of crossing cellular membranes (31, 32). Thus, we presume that the lack of inhibitory activity of -OONO is due to an inherent lack of reactivity with Ace1 and not a lack of accessibility. Also, it should be realized that most previous studies indicating that -OONO is capable of altering protein function via oxidation of crucial thiol residues were performed under in vitro conditions (for example, refs 28-30). Thus, intracellular thiol repair or -OONO scavenging systems were not available. In the study presented herein, these factors may also contribute to the lack of potency of -OONO as an inhibitor of Ace1. The O2-dependency in the NO-mediated inhibition of Ace1 can be explained alternatively by two other mechanisms. It is well established that NO can react with O2 to give NO2, which reacts further with NO to generate N2O3 (reactions 1 and 2). N2O3 is a nitrosating species capable of reacting with cellular nucleophiles, such as thiols, leading to oxidized species (reaction 5). It has also been proposed that NO is capable of reacting directly with thiols to give an intermediate radical anion species which is then capable of reducing O2 to O2- (reactions 8 and 9) (33).
RSH + NO f [RSNO]•- + H+
(8)
[RSNO]•- + O2 f RS-NO + O2-
(9)
Thus, thiol modification can presumably occur via NO/ O2 chemistry by generation either of N2O3 or from reactions 8 and 9. Both processes would be consistent with the observed O2-dependence for NO-mediated inhibition of Ace1. To distinguish between these two mech-
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anisms, we examined NO-mediated Ace1 inhibition under a variety of conditions. If N2O3 were responsible for the observed inhibitory activity of NO under aerobic conditions, oxidation of NO to NO2 via an alternative, O2independent mechanism should also cause inhibition. Fortunately, there exists an ideal reagent for performing these studies. The nitronyl nitroxide PTIO is capable of directly converting NO to NO2 in an O2-independent manner (34) and has even been utilized as an NO scavenger in animal models of endotoxin shock (35). The PTIO-dependent formation of NO2 from NO should lead to the in situ formation of N2O3 since the NO2 produced has the opportunity to react with excess NO (reaction 2). Indeed, we find that the combination of PTIO and NO is capable of inhibiting Ace1 activity under anaerobic conditions (Figure 3). Since much of the conversion of NO to NO2 may occur in the culture media, the generation of intracellular N2O3 may be dependent, in part, on the ability of NO and NO2 to cross the cellular membrane. Clearly, NO is a freely diffusable species (which is partly the basis for much of its biological affects). Significantly, NO2 will also traverse cell membranes as evidenced by its ability to cause damage to intracellular macromolecules, such as DNA, in intact cell preparations (for example, see ref 36). Thus, both NO and NO2 will traverse cell membranes and will be capable of intracellular N2O3 generation. It appears that the O2-dependence of the NO-mediated inhibition of copper-dependent Ace1 activity is due to the oxidation of NO to NO2 and not via the sequential reactions 8 and 9. However, it remains possible that the ultimate inhibitory species is not N2O3, but rather NO2 alone. Significantly, it has been reported that NO2 itself is capable of oxidizing thiols (23). To determine whether NO2 was the ultimate inhibitory species or whether formation of products derivative of NO2 (i.e., N2O3) were responsible for Ace1 modification, we examined the inhibitory activity of authentic NO2 gas. Thus, yeast were exposed to authentic NO and/or NO2 gases (added to the incubation headspace) under anaerobic conditions. Yeast exposure to these gases was either sequential or simultaneous. In this way, we were able to determine whether the maximum inhibition of Ace1 activity is observed only when yeast are exposed simultaneously to a combination of NO and NO2 or when exposed to them individually. As shown in Figure 4, the maximum inhibition of copperdependent Ace1 activity was observed when NO and NO2 were present simultaneously, although some inhibition did occur in the presence of NO2 alone. The observed NO2mediated inhibition may be due to thiol oxidation by NO2, as mentioned above, or the nitrosating ability of N2O4 (37). Regardless, the observed maximum inhibition exhibited by the simultaneous exposure to NO and NO2 is consistent with N2O3 generation as being responsible for much of the observed Ace1 inhibition. The majority of the observed inhibition of Ace1 activity by NO appears to be due to the generation of the nitrosating species N2O3. As mentioned previously, in situ generation of N2O3 can also be accomplished via acidification of NO2- (reactions 6 and 7) (38). Since the generation of N2O3 from NO2- is second order in HONO concentration (38), significant production of N2O3 will only occur at relatively high concentrations of NO2- and at relatively low pH. Indeed, this is the case as Figure 5
Shinyashiki et al.
shows that NO2- only inhibits Ace1 at high concentration and at low pH. Many of the studies performed herein were possible due to the versatility and well-defined nature of the yeast model system. For example, we were able to examine the effect of O2 and pH on nitrogen oxide-mediated events in order to elucidate the nature of the ultimate chemical reactant responsible for the biological effect. These types of experiments are not possible in mammalian cell culture. Thus, yeast is proving to be a powerful model system whereby the nature of the chemistry between nitrogen oxides and selected proteins can be delineated. Although there has yet to be identified a functional homologue for Ace1 in mammalian systems, an examination of its chemical interactions with nitrogen oxide species does serve as a basis for understanding similar biochemical events with other, structurally related mammalian proteins. For example, metallothionein (39, 40), mammalian copper chaperones (41-45), and zinc-finger proteins (46) all bind metals via thiolate ligation and may all interact with NO, and related nitrogen oxides, in a homologous manner. Our studies indicate that N2O3 is the most potent disruptor of copper-thiolate complexes among all accessible nitrogen oxide species derived from NO. Since the autoxidation of NO in aqueous solution is kinetically second order in NO (reaction 1) (47), significant N2O3 formation will only occur at relatively high concentrations of NO. Thus, our observation that N2O3 will be the primary NO-derived nitrogen oxide reactant with biological thiolate-metal complexes (in aerobic, aqueous solution) indicates that these proteins will only be disrupted in instances of high NO output. However, it should be noted that Liu and co-workers (48) have shown that biological N2O3 chemistry is much more accessible in lipophilic environments (i.e., membranes) indicating that membrane-bound metal-thiolate complexes may be particularly susceptible.
Acknowledgment. We would like to thank Dr. Dennis Thiele, Dr. Joan Valentine, and Dr. Edith Gralla for their valuable contribution to this work. This work was funded in part by a grant from the National Science Foundation (0096380) and supported in part by USPHS National Research Service Award GM08496.
References (1) Ignarro, L. J. (1989) Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ. Res. 65, 1-21. (2) Hobbs, A. J. (1997) Soluble guanylate cyclase: the forgotten sibling. Trends Pharmacol. Sci. 18, 484-491. (3) MacMicking, J., Xie, Q. W., and Nathan, C. (1997) Nitric oxide and macrophage function. Annu. Rev. Immunol. 15, 323-350. (4) Bouton, C. (1999) Nitrosative and oxidative modulation of iron regulatory proteins. Cell. Mol. Life Sci. 55, 1043-1053 (5) Kroncke, K. D., and Carlberg, C. (2000) Inactivation of zinc finger transcription factors provides a mechanism for a gene regulatory role of nitric oxide. FASEB J. 14, 166-173. (6) Shinyashiki, M., Chiang, K. T., Switzer, C. H., Gralla, E. B., Valentine, J. S., Thiele, D. J., and Fukuto, J. M. (2000) The interaction of nitric oxide (NO) with the yeast transcription factor Ace1: A model system for NO-protein thiol interactions with implications to metal metabolism. Proc. Natl. Acad. Sci. U.S.A. 97, 2491-2496. (7) Chiang, K. T., Shinyashiki, M., Switzer, C. H., Valentine, J. S., Gralla, E. B., Thiele, D. J., and Fukuto, J. M. (2000) Effects of nitric oxide on the copper-responsive transcription factor Ace1 in Saccharomyces cerevisiae: cytotoxic and cytoprotective actions of nitric oxide. Arch. Biochem. Biophys. 377, 296-303.
Mechanism of Ace1 Inhibition by NO (8) Askwith, C., and Kaplan, J. (1998) Iron and copper transport in yeast and its relevance to human disease. Trends Biochem. Sci. 23, 135-138. (9) Eide, D. J. (1998) The molecular biology of metal ion transport in Saccharomyces cerevisiae. Annu. Rev. Nutr. 18, 441-469. (10) Winge, D. R. (1998) Copper-regulatory domain involved in gene expression. Prog. Nucleic Acid Res. Mol. Biol. 58, 165-195. (11) Labbe, S., and Thiele, D. J. (1999) Pipes and wiring: the regulation of copper uptake and distribution in yeast. Trends Microbiol. 7, 500-505. (12) Santoro, N., Johansson, N., and Thiele, D. J. (1998) Heat shock element architecture is an important determinant in the temperature and transactivation domain requirements for heat shock transcription factor. Mol. Cell. Biol. 18, 6340-6352. (13) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. (14) Beckman, J. S, Chen, J., Ischiropoulos, H., and Crow, J. P. (1994) Oxidative chemistry of peroxynitrite. Methods Enzymol. 233, 229-240. (15) Huie, R. E., and Padmaja, S. (1993) The reaction of NO with superoxide. Free Radic. Res. Commun. 18, 195-199. (16) Goldstein, S., and Czapski, G. (1995) The reaction of NO. with O2•- and HO2•: a pulse radiolysis study. Free Radical Biol. Med. 19, 505-510. (17) Pryor, W. A., and Squadrito, G. L. (1995) The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. Am. J. Physiol. 268, L699-L722. (18) Akaike, T., Yoshida, M., Miyamoto, Y., Sato, K., Kohno, M., Sasamoto, K., Miyazaki, K. Ueda, S., and Maeda, H. (1993) Antagonistic action of imidazolineoxyl N-oxides against endothelium-derived relaxing factor/•NO through a radical reaction. Biochemistry 32, 827-832. (19) Jensen, L. T., Howard, W. R., Strain, J. J., Winge, D. R., and Culotta, V. C. (1996) Enhanced effectiveness of copper ion buffering by CUP1 metallothionein compared with CRS5 metallothionein in Saccharomyces cerevisiae. J. Biol. Chem. 271, 18514-18519. (20) Tamai, K. T., Gralla, E. B., Ellerby, L. M., Valentine, J. S., and Thiele, D. J. (1993) Yeast and mammalian metallothioneins functionally substitute for yeast copper-zinc superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 90, 8013-8017. (21) Dameron, C. T., Winge, D. R., George, G. N., Sansone, M., Hu, S., and Hamer, D. (1991) A copper-thiolate polynuclear cluster in the ACE1 transcription factor. Proc. Natl. Acad. Sci. U.S.A. 88, 6127-6131. (22) Dameron, C. T., George, G. N., Arnold, P., Santhanagopalan, V., and Winge, D. R. (1993) Distinct metal binding configurations in ACE1. Biochemistry 32, 7294-7301. (23) Prutz, W. A., Monig, H., Butler, J., and Land, E. J. (1985) Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins. Arch. Biochem. Biophys. 243, 125-134. (24) Radi, R., Beckman, J. S., Bush, K. M., and Freeman, B. A. (1991) Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266, 4244-4250. (25) Wink, D. A., Nims, R. W., Darbyshire, J. F., Christodoulou, D., Hanbauer, I., Cox, G. W., Laval, F., Laval, J., Cook, J. A., Krishna, M. C., DeGraff, W. G., and Mitchell, J. B. (1994) Reaction kinetics for nitrosation of cysteine and glutathione in aerobic nitric oxide solutions at neutral pH. Insights into the fate and physiological effects of intermediates generated in the NO/O2 reaction. Chem. Res. Toxicol. 7, 519-525. (26) Kharitonov, V. G., Sundquist, A. R., and Sharma, V. S. (1995) Kinetics of nitrosation of thiols by nitric oxide in the presence of oxygen. J. Biol. Chem. 270, 28158-28164. (27) Goldstein, S., and Czapski, G. (1996) Mechanism of the nitrosation of thiols and amines by oxygenated NO solutions: the nature of the nitrosating intermediates. J. Am. Chem. Soc. 118, 3419-3425.
Chem. Res. Toxicol., Vol. 14, No. 12, 2001 1589 (28) Kuhn, D. M., and Geddes, T. J. (1999) Peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. J. Biol. Chem. 274, 29726-29732. (29) Viner, R. I., Williams, T. D. and Schoneich, C (1999) Peroxynitrite modification of protein thiols: Oxidation, nitrosylation and Sglutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase. Biochemistry 38, 1240812415. (30) Crow, J. P., Beckman, J. S., and McCord, J. M. (1995) Sensitivity of the essential Zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite. Biochemistry 34, 35443552. (31) Marla, S. S., Lee, J., and Groves, J. T. (1997) Peroxynitrite rapidly permeates phospholipid membranes. Proc. Natl. Acad. Sci. U.S.A. 94, 14243-14248. (32) Denicola, A., Souza, J. M., and Radi, R. (1998) Diffusion of peroxynitrite across erythrocyte membranes. Proc. Natl. Acad. Sci. U.S.A. 95, 3566-3571. (33) Gow, A. J., Buerk, D. G., and Ischiropoulos, H. (1997) A novel reaction mechanism for the formation of S-nitrosothiol in vivo. J. Biol. Chem. 272, 2841-2845. (34) Akaike, T., and Maeda, H. (1996) Quantitation of nitric oxide using 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Methods Enzymol. 268, 211-221. (35) Yoshida, M., Akaike, T., Wada, Y., Sato, K., Ikeda, K. Ueda, S., and Maeda, H. (1994) Therapeutic effects of imidazolineoxyl N-oxide against endotoxin shock through its direct nitric oxidescavenging activity. Biochem. Biophys. Res. Commun. 202, 923930. (36) Bittrich, H., Matzig, A. K., Kraker, I., and Appel, K. E. (1993) NO2-induced DNA single strand breaks are inhibited by antioxidative vitamins in V79 cells, Chem.-Biol. Interact. 86, 199-211. (37) Challis, B. C., and Kyrtopoulos, S. A. (1978) The chemistry of nitroso compounds. Part 12. The mechanism of nitrosation and nitration of aqueous piperidine by gaseous dinitrogen tetraoxide and dinitrogen trioxide in aqueous alkaline solutions. Evidence for the existence of molecular isomers of dinitrogen tetraoxide and dinitrogen trioxide. J. Chem. Soc., Perkin Trans 2, 12961302. (38) Williams, D. L. H. (1983) Nitrosation mechanisms. Adv. Physiol. Organic Chem. 19, 381-429. (39) Boulanger, Y., and Armitage, I. M. (1982) 113Cd NMR study of the metal cluster structure of human liver metallothionein. J. Inorg. Biochem. 17, 147-153. (40) Boulanger, Y., Armitage, I. M., Miklossy, K. A., and Winge, D. R. (1982) 113Cd NMR study of a metallothionein fragment. Evidence for a two-domain structure. J. Biol. Chem. 257, 13717-13719. (41) Vulpe, C. D., and Packman, S. (1995) Cellular copper transport. Annu. Rev. Nutr. 15, 293-322. (42) Eide, D. (1997) Molecular biology of iron and zinc uptake in eukaryotes. Curr. Opin. Cell Biol. 9, 573-577. (43) Valentine, J. S., and Gralla, E. B. (1997) Delivering copper inside yeast and human cells. Science 278, 817-818. (44) Winzerling, J. J., and Law, J. H. (1997) Comparative nutrition of iron and copper. Annu. Rev. Nutr. 17, 501-526. (45) Askwith, C., and Kaplan, J. (1998) Iron and copper transport in yeast and its relevance to human disease. Trends Biochem. Sci. 23, 135-138. (46) Vallee, B. L., and Falchuk, K. H. (1993) The biochemical basis of zinc physiology. Physiol. Rev. 73, 79-118. (47) Ford, P. C., Wink, D. A., and Stanbury, D. M. (1993) Autoxidation kinetics of aqueous nitric oxide. FEBS Lett. 326, 1-3. (48) Liu, X., Miller, M. J., Joshi, M. S., Thomas, D. D., and Lancaster, J. R., Jr. (1998) Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes. Proc. Natl. Acad. Sci. U.S.A. 95, 2175-2179.
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