Reconstitution of apo-superoxide dismutase by nitric oxide-induced copper transfer from metallothioneins

Little is known about copper transfer from Cu-metallothionein (Cu-MT) to va rious target proteins, such as apo-SOD, and the potential role of redox mec hanisms in this transfer. We studied Cu transfer from Cu-MT to apo/Zn-SOD i n a cell-free model system and found that Cu-5-MT and Cute-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 Cu-5-MT and Cu-10-MT was 34 and 83%, re spectively, compared with that reconstituted by free Cu alone. A Cu chelati on assay using bathocuproine disulfonate (BCS) showed that NOC-15 induced r elease of free Cu from Cute-MT but not from Cu-5-MT. The transfer of Cu fro m Cu-MT to apo/Zn-SOD was not accompanied by enhanced Cu-dependent generati on of ascorbate radicals or hydroxyl radicals as measured by EPR spectrosco py. We found a 70% decrease in the number of 2,2'-dithiodipyridine titratab le SH groups on MT after incubation with NOC-15. Overall, our results sugge st that Cu-MT could potentially function in a nitric oxide-dependent pathwa y for the delivery of Cu to apo-SOD in copper-challenged cells.