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Chem. Res. Toxicol. 2000, 13, 922-931
Reconstitution of Apo-Superoxide Dismutase by Nitric Oxide-Induced Copper Transfer from Metallothioneins Shang-Xi Liu,† James P. Fabisiak,† Vladimir A. Tyurin,†,‡ Grigory G. Borisenko,† Bruce R. Pitt,§ John S. Lazo,§ and Valerian E. Kagan*,†,§ Department of Environmental & Occupational Health, Graduate School of Public Health, and Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15238 Received March 20, 2000
Little is known about copper transfer from Cu-metallothionein (Cu-MT) to various target proteins, such as apo-SOD, and the potential role of redox mechanisms in this transfer. We studied Cu transfer from Cu-MT to apo/Zn-SOD in a cell-free model system and found that Cu5-MT and Cu10-MT were able to reconstitute SOD activity only in the presence of a nitric oxide donor, (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (NOC-15). The percentage of reconstitution by Cu5-MT and Cu10-MT was 34 and 83%, respectively, compared with that reconstituted by free Cu alone. A Cu chelation assay using bathocuproine disulfonate (BCS) showed that NOC-15 induced release of free Cu from Cu10-MT but not from Cu5-MT. The transfer of Cu from Cu-MT to apo/Zn-SOD was not accompanied by enhanced Cu-dependent generation of ascorbate radicals or hydroxyl radicals as measured by EPR spectroscopy. We found a 70% decrease in the number of 2,2′-dithiodipyridine titratable SH groups on MT after incubation with NOC-15. Overall, our results suggest that Cu-MT could potentially function in a nitric oxide-dependent pathway for the delivery of Cu to apo-SOD in copper-challenged cells.
Introduction A number of environmental sources may contribute to copper overload, including water contaminated from surrounding mines or from copper-contaminated untinned pipes, fly ash residue produced by heat treatment of municipal solid wastes, and waste byproducts generated by poultry industry as a result of feeding birds with the excess trace elements (including copper) (1, 2). As a transition metal, copper participates in Fenton-type reactions to yield several reactive oxygen species (3). The Cu+-Cu2+ redox couple can reduce oxygen to superoxide, whose dismutation produces hydrogen peroxide. The latter is further decomposed by Cu(I) to generate hydroxyl radical capable of initiating damage to nucleic acids, proteins, and lipids. Copper, however, is an essential metal necessary for the function of several enzymes, including cytochrome oxidase, tyrosinase, and Cu/Zn superoxide dismutase (Cu/Zn-SOD).1 Thus, the intracellular trafficking of Cu must be tightly controlled to avoid its injurious redox cycling (4). Indeed, Rae et al. (5) have shown that yeast contains less than one single free copper ion/cell under basal growth conditions. Recently, three Cu chaperones, Atx1 (6), Cox17 (7), and CCS (8), that safely deliver Cu to specific intracellular * To whom correspondence should be addressed: Department of Environmental and Occupational Health, 260 Kappa Dr., University of Pittsburgh, Pittsburgh, PA 15238. Telephone: (412) 967-6516. Fax: (412) 624-1020. E-mail:
[email protected]. † Department of Environmental & Occupational Health, Graduate School of Public Health, University of Pittsburgh. ‡ On leave from the Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg 194223, Russia. § Department of Pharmacology, School of Medicine, University of Pittsburgh.
locations at low copper concentrations have been characterized. However, the role of the chaperones in copper delivery at high copper levels is negligible (5-8). Importantly, metallothioneins (MTs), low-molecular mass (approximately 6000 Da) proteins, act as major intracellular Cu-binding proteins especially in the presence of excess Cu (9-12). Being cysteine-rich proteins, MTs readily release metals in a redox-dependent fashion. For example, nitrosylation of MT cysteines has been shown to release MTbound cadmium and enhance cytotoxicity of this metal (13). Furthermore, redox-driven delivery of zinc from MT to target proteins has recently been established and suggested to establish a physiological role for MT in metal trafficking (14-16). Cu/Zn-SOD is a homodimeric enzyme that catalyzes the dismutation of superoxide radical through the cyclic reduction and oxidation of active site copper and produces molecular oxygen and hydrogen peroxide (17, 18). The nascent form of SOD synthesized by the cell is Cu- and Zn-free apo-SOD (19, 20), and the exact method by which apo-SOD obtains Cu to become the active enzyme without compromising redox safety is still not clear. CCS (copper chaperone for SOD) functions as an important Cu delivery mechanism to apo-SOD in yeast at very low Cu concentrations (5). A significant pool of apo1 Abbreviations: BCS, bathocuproine disulfonic acid, disodium salt; CCS, copper chaperone for SOD; Cu/Zn-SOD, Cu/Zn superoxide dismutase; DDC, diethyl dithiocarbamate; DMPO, 5,5-dimethyl-1pyrroline N-oxide; DTDP, 2,2′-dithiodipyridine; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; MT, metallothionein; NBT, nitro blue tetrazolium; NOC-15, (Z)-[N-(3ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate; N-PAGE, native polyacrylamide gel electrophoresis; PMS, phenazine metasulfate.
10.1021/tx0000623 CCC: $19.00 © 2000 American Chemical Society Published on Web 08/19/2000
Copper Delivery by Metallothioneins
SOD (35% of total), however, exists in lymphoblastoid, K562, and HL-60 cells grown under normal conditions (21-23). While the size of this pool of apo-SOD decreased in the presence of excess Cu, it was not fully eliminated even after 15 h incubations (21). Thus, the extent of delivery of Cu to apo-SOD cannot be solely determined by the availability of free Cu. Importantly, increased Cu/Zn-SOD activity can be induced by oxidative stress in the absence of transcriptional activation (22, 24, 25), suggesting that redox-induced transfer of copper to apo-SOD could account for acute regulation of SOD enzyme activity. We have previously shown that MT binding and release of Cu can be regulated by oxidation of MT cysteines (26) and MT binding capacity could be regenerated by cysteine recycling (26). Since release of MT-bound Cu from thiolate clusters is redox-sensitive, but binding to His46, His48, His63, and His120 Cu coordination sites of apo-SOD is not redox-dependent (27, 28), the MT-apoSOD couple is an ideal candidate for copper donoracceptor interactions. We speculate that in Cu-challenged cells, Cu bound in MT could be released by oxidizing conditions, be incorporated into apo-SOD, and thus activate the SOD enzyme. Here we studied Cu-MT-apoSOD interactions in a cell-free model system and identified redox conditions providing for the efficient transfer of Cu to apo-SOD and reconstitution of enzymatic activity without deleterious release of free Cu.
Materials and Methods Reagents. Zn-MT-1, isolated from rabbit liver, was kindly provided by D. Petering (University of Wisconsin, Milwaukee, WI). Cu/Zn-SOD purified from bovine erythrocytes (5100 units/ mg of protein), diethyl dithiocarbamate (DDC), CuSO4, sodium ascorbate, diphenylthiocarbazone (dithizone), nitro blue tetrazolium (NBT), β-NADH, phenazine metasulfate (PMS), and riboflavin were from Sigma Chemical Co. (St. Louis, MO). 5,5Dimethyl-1-pyrroline N-oxide (DMPO) was purchased from Aldrich (Milwaukee, WI) and bathocuproine disulfonic acid, disodium salt (BCS), from Fisher Scientific (Pittsburgh, PA). The NO donor, NOC-15 {(Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate}, was purchased from Alexis Biochemicals (San Diego, CA), and a concentrated stock solution was freshly prepared by dissolution in 20 mM NaOH. To remove trace metal ions, all the buffer solutions used in the experiments were treated with dithizone (29). Preparation of Apo/Zn-SOD. Apo/Zn-SOD was prepared by the method reported by Cocco et al. (30). Using this method, more than 90% of the Cu in SOD is removed with complete retention of Zn. Briefly, DDC was added to a Cu/Zn-SOD (0.1 mM) solution in 0.1 M potassium phosphate (pH 7.4) at a ratio of 10:1. The mixture was incubated at 37 °C for 3 h. The solution containing the yellow DDC-Cu complex was then centrifuged at 39000g for 30 min. The resulting colorless supernatant was then exhaustively dialyzed against the phosphate buffer and stored at 4 °C. Preparation of Cu-MT. Cu-MT was prepared as previously described (26). We incubated Zn7-MT with CuSO4 (200 µM) in the presence of ascorbate (800 µM). Ascorbate was included to maintain a steady-state concentration of Cu+, the form of Cu bound by MT. Zn-MT was included at two concentrations corresponding to Cu-MT molar ratios of 5:1 and 10:1 relative to CuSO4. This was done to yield MT preparations that include partially saturated Cu5-MT and Cu10-MT species of Cu-MT. After incubation for 20 min at room temperature, the Cu-MTs were directly used in the experiments for reconstitution of apo/ Zn-SOD. Reconstitution of Apo/Zn-SOD by Cu-MT. Apo/Zn-SOD (10 µM) was incubated with either Zn7-MT, Cu5-MT, or Cu10MT at a molar ratio of 1:1 in 50 mM phosphate buffer (pH 7.4)
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 923 in the presence or absence of NOC-15 (1 mM) at room temperature for 2 h. At the end of the incubation, ethylenediaminetetraacetic acid (EDTA) (0.1 mM, final concentration) was added to prevent further reconstitution. SOD Activity Assay. SOD activity was measured using the microtiter plate method reported by Ewing and Janero (31). For the assay, 25 µL samples were pipetted into a microtiter well containing 200 µL of freshly prepared 0.1 mM EDTA, 62 µM NBT, and 98 µM NADH in 50 mM phosphate buffer (pH 7.4). The reaction was initiated by the addition of 25 µL of freshly prepared 33 µM PMS in 50 mM phosphate buffer (pH 7.4), containing 0.1 mM EDTA. The absorbance at 600 nm was measured at the start of the reaction and again after incubation for 5 min at room temperature using a Dynatech MRX microtiter plate reader. One unit of SOD was defined as the activity required to inhibit the initial rate of NBT reduction by 50% (IC50). Protein was quantified by the Bradford dye binding assay as described by the manufacturer’s instructions (Bio-Rad, Richmond, CA). Polyacrylamide Gel Electrophoresis. Native polyacrylamide gel electrophoresis (N-PAGE) was performed by the method of Sambrook using an 8% acrylamide gel (32). Gels were stained either for protein with Coomassie Blue (R-250) or for enzyme activity as described by Beauchamp and Fridovich (33) in a solution containing 50 mM potassium phosphate (pH 7.8), 336 µM NBT, 172 µM riboflavin, and 21 mM TEMED. After incubation for 45 min in the dark, the blue NBT stain indicating superoxide anion was developed by exposure to light. BCS/Ascorbate Assay of Free and “Loosely Bound” Cu in Cu-MT. Samples from Cu-MT incubations with or without NOC-15 were added to a BCS (40 µM) and ascorbate (100 µM) solution in 10 mM Tris-HCl buffer (pH 7.4) to obtain a final Cu concentration of
