Metallothioneins (MTs) are cysteine-rich, metal-binding proteins that are found throughout Nature. This ubiquity highlights their importance in essential metal regulation, heavy metal detoxification and cellular redox chemistry. Missing from the current description of MT function is the underlying mechanism by which MTs achieve their proposed biological functions. To date, there have been conflicting reports on the mechanism of metal binding and the structures of the metal binding intermediates formed during metalation of apoMTs. The form of the metal-bound intermediates dictates the metal sequestering and metal-donating properties of the protein. Through a detailed analysis of spectral data from electrospray ionization mass spectromeric and circular dichroism methods we report that Zn(ii) and Cd(ii) metalation of the human MT1a takes place through two distinct pathways. The first pathway involves formation of beaded structures with up to five metals bound terminally to the 20 cysteines of the protein via a noncooperative mechanism. The second pathway is dominated by the formation of the four-metal domain cluster structure M4SCYS11via a cooperative mechanism. We report that there are different pathway preferences for Zn(ii) and Cd(ii) metalation of apo-hMT1a. Cd(ii) binding follows the beaded pathway above pH 7.1 but beginning below pH 7.1 the clustered (Cd4Scys11) pathway begins to dominate. In contrast, Zn(ii) binding follows the terminal, "beaded", pathway at all physiologically relevant pH (pH ≥ 5.2) only following the clustered pathway below pH 5.1. The results presented here allow us to reconcile the conflicting reports concerning the presence of different metalation intermediates of MTs. The conflict regarding cooperative versus noncooperative binding mechanisms is also reconciled with the experimental results described here. These two metal-specific pathways and the presence of radically different intermediate structures provide insight into the multi-functional nature of MT: binding Zn(ii) terminally for donation to metalloenzymes and sequestering toxic Cd(ii) in a cluster structure.

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.

Metallothioneins (MTs) are small, cysteine-rich proteins, found throughout Nature. Their ability to bind a number of different metals with a range of stoichiometric ratios means that this protein family is critically important for essential metal (Zn and Cu) homeostasis, metal storage, metal donation to nascent metalloenzymes as well as heavy metal detoxification. With its 20 cysteines, metallothionein is also considered to protect cells against oxidative stress. MT has been studied by a large number of researchers over the last 6 decades using a variety of spectroscopic techniques. The lack of distinguishing chromophores for the multitude of binding sites has made the evaluation of stoichiometric properties for different metals challenging. Initially, only Cd-NMR spectroscopy could provide strong evidence for the proposed cluster formation of Cd-MT. The extraordinary development of electrospray ionization mass spectrometry (ESI-MS), where all coexisting species in solution are observed, revolutionized MT research. Prior to the use of ESI-MS data, a range of "magic numbers" representing metal-to-MT molar ratios were reported from optical spectroscopic studies. The availability of ESI mass spectral data led to (i) the confirmation of cluster formation, (ii) a conceptual understanding of the cooperativity involved in multiple metal binding events, (iii) the presence of domain specificity between regions of the protein and (iv) mechanistic details involving both binding affinities and rate constants. The kinetic experiments identified the presence of multiple individual binding sites, each with a unique rate constant and an analogous binding affinity. The almost linear trend in rate constants as a function of bound As provided a unique insight that became a critical step in the complete understanding of the mechanistic details of the metalation of MT. To fully define the biological function of this sulfur-rich protein it is necessary to determine kinetic rate constants and binding affinities for the essential metals. Recently, Zn competition experiments between both of the isolated fragments (α and β) and the full-length protein (βα-MT 1a) as well as Zn competition between βα-MT 1a and carbonic anhydrase were reported. From these data, the trend in binding affinities and the values of the K of the 7 bimolecular reactions involved in metalation were determined. From the analysis of ESI-MS data for Cu binding to βα-MT 1a at different pH-values, a trend in the 20 binding affinities for the complete metalation mechanism was reported. This review details a personal view of the historical development of the determination of stoichiometry for metal binding, the structure of the binding sites, the rates of the metalation reactions and the underlying binding affinities for each metalation step. We have attempted to summarize the experimental developments that led to the publication in May 2017 of the experimental determination of the 20 binding constants for the 20 sequential bimolecular reactions for Cu binding to the 20 Cy...

Human metallothionein 1a, a protein with two cysteine-rich metal-binding domains (α with 11 Cys and β with 9), was analyzed in its metal-free form by selective, covalent Cys modification coupled with ESI-MS. The modification profiles of the isolated β- and α-fragments reacted with p-benzoquinone (Bq), N-ethylmalemide (NEM) and iodoacetamide (IAM) were compared with the full length protein using ESI-mass spectral data to follow the reaction pathway. Under denaturing conditions at low pH, the reaction profile with each modifier followed pathways that resulted in stochastic, Normal distributions of species whose maxima was equal to the mol. eq. of modifier added. Our interpretation of modification at this pH is that reaction with the cysteines is unimpeded when the full protein or those of its isolated domains are denatured. At neutral pH, where the protein is expected to be folded in a more compact structure, there is a difference in the larger Bq and NEM modification, whose reaction profiles indicate a cooperative pattern. The reaction profile with IAM under native conditions follows a similar stochastic distribution as at low pH, suggesting that this modifier is small enough to access the cysteines unimpeded by the compact structure. The data emphasize the utility of residue modification coupled with electrospray ionization mass spectrometry for the study of protein structure.