Mobility of Copper in Binding Sites in Rabbit Liver Metallothionein 2

Abstract

We describe the first direct evidence for mobility of Cu(I) atoms within the metal binding sites in mammalian metallothionein based on temporal changes in the emission spectrum of Cun-MT (n = 1−20) in the 600 nm region. The emission intensities are specifically dependent on the temperature and on the metal:protein molar ratio between 1 Cu(I), 12 Cu(I), and 20 Cu(I) [Green, A. R.; Presta, A. P.; Gasyna, Z.; Stillman, M. J. Inorg. Chem. 1994, 33, 4159−4168]. We report that this emission spectral intensity systematically changes with time in the period following the initial binding of Cu(I) to rabbit liver Zn7-MT that can be interpreted on a molecular level in terms of the adoption by Cu(I) of different copper−thiolate cluster structures within the binding site over a period of 18 min (at room temperature) following initial binding of the Cu(I) atoms. The kinetic traces show three significantly different trends depending on the Cu(I):MT ratio. Quantitative analysis shows that the rate of this reaction slows considerably when Cu−Scys−Cu bridges must form. After an initial rise in the emission intensity during the first 5 min, the emission intensity either (i) decreases (1−8 Cu(I)), (ii) increases (9−12 Cu(I)), or (iii) remains constant (13−20 Cu(I)). The latter two trends in particular confirm the stability of the copper(I)-containing protein over time. Both qualitative and quantitative interpretation show that the final structure adopted is not the same as that formed immediately after the Cu(I) binds to the thiolate groups in Zn-MT for all metal loading ratios between 1 and 12 Cu(I). The data confirm that Cu(I) initially binds to Zn7-MT in both the α and β domains in a noncooperative, random manner. After equilibration, the Cu(I) atoms rearrange to fill the less emissive β domain preferentially. This rearrangement is similar to that observed for Cd(II) during the formation of Cd4-MT α-fragment when Cd(II) is added to Zn7-MT [Stillman, M. J.; Zelazowski, A. J. Biochem. J. 1989, 262, 181−188] and accounts for the decrease in emission with time as 1−8 Cu(I) are added to Zn7-MT. The data for 1−12 Cu(I) can be interpreted on a molecular level in terms of the mobility of bound Cu(I) between different sites in the protein and in terms of the flexibility of the peptide chain providing the first direct spectroscopic evidence that Cu(I) atoms migrate between thiolate cluster sites following initial binding. The Cu(I) bound in the α domain in the kinetically-controlled domain-distributed product migrates to the β domain to form the domain-specific thermodynamically-controlled product. The data thus provide evidence for the origin of the previously reported domain specificity for Cu(I) binding to Zn-MT. Further rearrangements by the peptide backbone account for the increase in emission intensity with time as 9−12 Cu(I) are added to Zn7-MT.