Emission spectral data measured over a range of temperatures are reported for copper(I) binding to aqueous solutions of rabbit liver zinc metallothionein at pH 7. These data provide a unique probe of the pathways adopted as copper-thiolate clusters form in the metallothionein. Metal analysis shows that, at low temperatures (< 15-degrees-C), Cu(I) displaces the Zn(II) linearly as a function of [Cu(I)] until 12 Cu(I) have been added, at which point all 7 Zn(II) are displaced. At high temperatures, significant hysteresis in the displacement of the Zn(II) results in nonlinearity in the [Zn(II)] vs [Cu(I)] line at the 6 Cu(I) point. As Cu(I) is added to Zn7-MT an emission band near 600 nm intensifies at all temperatures. At low temperatures (0-degree-C < T < 15-degrees-C) the normalized intensity (emission intensity as a function of Cu(I) bound) increases roughly linearly with a significant increase in emission when 12 Cu(I) are bound. At higher temperatures (15-degrees-C < T < 50-degrees-C) a completely different relationship between emission intensity and molar ratio of Cu(I) added to the Zn7-MT is observed. The normalized emission intensity decreases between 2 and 7 Cu(I) added. Between 7 and 12 Cu(I) added there is a dramatic increase in emission intensity. As at low temperatures, the emission intensity for 12 Cu(I) greatly exceeds 12 times the intensity for 1 Cu(I). The emission intensity decreases toward zero as from 13 to 20 Cu(I) are added at all temperatures. The band center of the copper-thiolate emission near 600 nm remains approximately constant until 11 Cu(I) have been added and then blue shifts sharply at the 12 Cu(I) point, before significantly red shifting by 20 nm between 13 and 16 Cu(I). The emission intensity changes dramatically following temperature cycles between low and high temperatures. These changes can only be interpreted in terms of the mobility of the Cu(I) bound to the protein. When between 1 and 7 Cu(I) are added to Zn7-MT at 6-degrees-C, the emission intensity is relatively high. Upon heating to 50-degrees-C, the emission intensity drops to 10% of this initial value and cooling the solution back to 6-degrees-C only recovers 40% of the original intensity. In complete contrast, for solutions containing between 8 and 12 Cu(I), the intensity after this same cycle is twice that of the original solution. The emission intensity clearly probes the location and structural features of the copper-thiolate clusters that form as up to 12 Cu(I) bind to the 20 cysteinyl thiolates in metallothionein. Through intrepretation of these experimental properties, the pathways by which individual Cu(I) atoms bind to Zn7-MT can be completely described on the basis of the following: (i) Cu(I) thiolate clusters in the alpha domain emit 4-10 times the light of Cu(I) in the beta domain. (ii) Cu(I) atoms located in the alpha domain can be detected by this high emission intensity. (iii) Cu(I) binds in a distributed manner statistically across both domains at all temperature. (iv) At high temperatures Cu(I) redistributes to populate the beta domain, forming the domain specific product, Cu6(S(cys))9-beta, which results in significant reduction in the emission intensity. (v) Cu(I) does not bind to Zn7-MT cooperatively. (vi) Cu12-MT is a tight structure, efficiently excluding solvent access to the Cu(I)-thiolate clusters. Finally, (vii) analysis of changes in the emission intensities as a function of temperature shows that these data provide a unique and sensitive probe of solvent access to the metal-thiolate clusters through the outer structure of the metal binding site. Analysis of the emission quenching suggests that the crevices predicted in the structure of Cu12-MT by molecular modeling techniques are present in Cu12-MT much like in the structure of Cd5Zn2-MT.
