OXIDATIVE QUENCHING OF LUMINESCENCE FROM COPPER-METALLOTHIONEIN

Mammalian metallothioneins readily bind up to 20 Cu(I); with a two domain structure involving Cu-6(S-cys)(11) (alpha) and Cu-6(S-cys)(9) (beta) copper-thiolate clusters proposed for the Cu-12-MT species. Copper metallothioneins luminesce in the 600 nm region when excited by 300 nm light. The 300 nm Stokes' shift and the dependence of the 600 nm emission band entirely on the presence of Cu(I) provides a sensitive probe of the copper-thiolate cluster structures that form the Cu(I) binding site in Cu,-MT, where n=1-12. Luminescence quenching data for the emissive Cu(I) state were used to monitor electron transfer in copper metallothionein. CD spectral data measured together with the emission spectrum show that the three-dimensional structure of the protein is maintained even when the excited state is completely quenched by molecular oxygen. Different acrylamide quenching rates for (Zn-4)(alpha)(Cu-6)(beta)-MT and (Cu-6)(alpha)(Cu-6)(beta)-MT suggest that structural differences dependent on the Cu(I) loading, especially the occupation of one or both domains by Cu(I), may influence the excited state properties of copper-containing mammalian metallothioneins. Finally, Fe(III) is efficiently reduced to Fe(II) following oxidative quenching of the luminescence of the Cu-12-MT; CD spectroscopy shows that the three-dimensional structure of Cu-12-MT is destroyed.