Cysteine accessibility during As3+ metalation of the α- and β-domains of recombinant human MT1a

Metallothionein is a ubiquitous metal binding protein that plays an important role in metal ion homeostasis and redox chemistry within cells. Mammalian metallothioneins bind a wide variety of metals including the metalloid As3+ in two domains (beta and alpha) connected by a short linker sequence. Three As3+ bind in each domain for a total of 6 As3+ per protein. In recombinant human metallothionein (rh-MT1a) each As3+ binds three cysteine residues to form As(3)Cys(9)(CysSH)(2)-alpha-rhMT1a in the 11 Cys alpha-domain and As(3)Cys(9)-beta-rhMT1a in the 9 Cys beta-domain. This means that there should be 2 free cysteines in the a-domain but no free cysteines in the beta-domain. By using benzoquinone, the number and relative accessibility of the free cysteinyl thiols during the metalation reactions were determined. The electrospray ionization mass spectrometry (ESI-MS) data confirmed that each As3+ binds using exactly 3 cysteine thiols and showed that there was a significant difference in the reactivity of the free cysteines during the metalation reaction. After a reaction with two molar equivalents of As3+ to form As(2)Cys(6)(CysSH)(3)-alpha beta-rhMT1a, the remaining 3 Cys in the 9 Cys beta-domain were far less reactive than those in the alpha-domain. Molecular dynamics calculations for the metalation reactions with As3+ measured by ESI-MS allowed an interpretation of the mass spectral data in terms of the relative location of the cysteine thiols that were not involved in As3+ coordination. Together, these data provide insight into the selection of a specific cysteinyl thiol by the incoming metals during the stepwise metalation of metallothioneins. (C) 2013 Elsevier Inc. All rights reserved.