Copper is a ubiquitous trace metal of vital importance in that it serves as a cofactor in many metalloenzymes. Excess copper becomes harmful if not sequestered appropriately in the cell. As a metal ion chaperone, metallothionein (MT) has been proposed as a key player in zinc and copper homeostasis within the cell. The underlying mechanisms by which MT sequesters and transfers copper ions, and subsequently achieves its proposed biological function remain unknown. Using a combination of electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and emission spectroscopy, we report that the Cu(i) to human apo-MT1a binding mechanism is highly pH-dependent. The 20 relative K_f-values for the binding of 1 to 20 Cu(i) to the 20 cysteines of MT were obtained from computational simulation of the experimental mass spectral results. These data identified the pH-dependent formation of three sequential but completely different Cu-S_CYS clusters, as a function of Cu(i) loading. These data provide the first overall sequence for Cu(i) binding in terms of domain specificity and transient binding site structures. Under cooperative binding at pH 7.4, a series of four clusters form: Cu₄S_CYS-6, followed by Cu₆S_CYS-9 (β), then a second Cu₄S_CYS-6 (α), and finally Cu₇S_CYS-x (α) (x = up to 11). Upon further addition of Cu(i), a mixture of species is formed in a non-cooperative mechanism, saturating the 20 cysteines of MT1a. Using benzoquinone, a cysteine modifier, we were able to confirm that Cu₆S_CYS-9 formed solely in the N-terminal β-domain, as well as confirming the existence of the presumed Cu₄S_CYS-6 cluster in the α-domain. Based on the results of ESI-MS and computational simulation we were able to identify Cu:MT speciation that resulted in specific emission and CD spectral properties.

Judith S. Scheller

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