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-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 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: CuS, followed by CuS (β), then a second CuS (α), and finally CuS (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 CuS formed solely in the N-terminal β-domain, as well as confirming the existence of the presumed CuS 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.

Metal-based therapeutics are vital tools in medicine. Metal-chelating proteins can dramatically decrease drug efficacy. Dirhodium(II) tetraacetate, a potential anticancer compound, binds in vitro to 8 cysteines of the human metallothionein 1a β-fragment. Electrospray ionization mass spectrometry shows that the final product is the Rh2(4+) core encapsulated by the β fragment of the metallothionein protein protein.

cis-Diamminedichloroplatinum(ii) (cisplatin), a powerful chemotherapeutic, can incur chemoresistance in cancers, reducing therapeutic success. Metallothioneins (MTs) are suspected of metallodrug interference via ligand removal and metal sequestration. The mechanistic details and reactions rates kobs for the systematic deconstruction of cisplatin by apo-human MT are reported and analysed from mass spectral data.

Anthropogenic sources of xenobiotic metals with no physiological benefit are increasingly prevalent in the environment. The platinum group metals (Pd, Pt, Rh, Ru, Os, and Ir) are found in marine and plant species near urban sources, and are known to bioaccumulate, introducing these metals into the human food chain. Many of these metals are also being used in innovative cancer therapy, which leads to a direct source of exposure for humans. This paper aims to further our understanding of nontraditional metal metabolism via metallothionein, a protein involved in physiologically important metal homeostasis. The aggressive reaction of metallothionein and dirhodium(II) tetraacetate, a common synthetic catalyst known for its cytotoxicity, was studied in detail in vitro. Optical spectroscopic and equilibrium and time-dependent mass spectral data were used to define binding constants for this robust reaction, and molecular dynamics calculations were conducted to explain the observed results.

The metallation of metallothionein can proceed via two different intermediate structures: a beaded structure that forms quickly (top) and a slow-forming cluster structure (bottom) before forming the fully metallated two-domain protein.