Reconstitution of Apo-Superoxide Dismutase by Nitric Oxide-Induced Copper Transfer from Metallothioneins

• Published in: Chemical research in toxicology 2000-09, Vol.13 (9), p.922-931
• Main Authors: Liu, Shang-Xi, Fabisiak, James P, Tyurin, Vladimir A, Borisenko, Grigory G, Pitt, Bruce R, Lazo, John S, Kagan, Valerian E
• Format: Article
• Language: English
• Subjects: Animals; Ascorbic Acid - metabolism; Azetidines - metabolism; Copper - metabolism; Electrophoresis, Polyacrylamide Gel; Hydroxyl Radical - metabolism; metallothionein; Metallothionein - metabolism; Molecular Chaperones - metabolism; Nitric Oxide - metabolism; Nitric Oxide Donors - metabolism; Rabbits; Superoxide Dismutase - metabolism; Zinc - metabolism
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx0000623

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.