TY - JOUR
T1 - Evidence of native metal-S(2-)-metallothionein complexes confirmed by the analysis of Cup1 divalent-metal-ion binding properties.
AU - Orihuela, Ruben
AU - Monteiro, Freddy
AU - Pagani, Ayelen
AU - Capdevila, Merce
AU - Atrian, Silvia
PY - 2010/11/2
Y1 - 2010/11/2
N2 - It has previously been shown that recombinant synthesis, under metal-supplemented conditions, of diverse metallothioneins (MTs) results in the recovery of a subpopulation of S2--containing complexes in addition to the S2--devoid canonical metal-MT species. Further significance of this finding has remained veiled by the possibility of it being a mere consequence of synthesis in a heterologous bacterial system. Herein, we present definitive evidence that S2- ligands are also constituents of native metal-MT complexes. Because, although practically universal, the highest S 2- content is incorporated by copper-thioneins when coordinating divalent metal ions, we adapted the Saccharomyces cerevisiae Cup1 protein, which is the most paradigmatic copper-thionein, as an experimental model. Most significantly, native Cd-Cup1 complexes were purified and fully spectroscopically and spectrometrically characterized from the 301N mutant yeast strain, which allows Cup1 synthesis even in the absence of copper. These results undoubtedly revealed the presence of a Cd-S2--Cup1 species in native preparations, which were only recovered when carefully avoiding the use of ion-exchange chromatography in the purification protocol. Furthermore, complete analysis of recombinant (Escherichia coli) Zn-Cup1, Cd-Cup1, and Cu-Cup1 and those complexes that result from Zn/Cd and Zn/Cu replacements in vitro and acidification/renaturalization processes yielded a comprehensive and comparative overview of the metal-binding abilities of Cup1. Overall, we consider the main conclusions of this study to go beyond the mere study of the particular Cup1 MT, so that they should be considered to delineate a new point of view on the interaction between copper-thioneins and divalent metal ions, still an unexplored aspect in MT research. Definitive evidence that sulfide ligands are also constituents of native metal-metallothionein (MT) complexes (see picture) is provided. Therefore, this investigation goes beyond the study of a particular MT and complements previous results about the presence of sulfide moieties in recombinant MT samples. A new point of view on the interaction between copper thioneins and divalent metal ions is delineated. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
AB - It has previously been shown that recombinant synthesis, under metal-supplemented conditions, of diverse metallothioneins (MTs) results in the recovery of a subpopulation of S2--containing complexes in addition to the S2--devoid canonical metal-MT species. Further significance of this finding has remained veiled by the possibility of it being a mere consequence of synthesis in a heterologous bacterial system. Herein, we present definitive evidence that S2- ligands are also constituents of native metal-MT complexes. Because, although practically universal, the highest S 2- content is incorporated by copper-thioneins when coordinating divalent metal ions, we adapted the Saccharomyces cerevisiae Cup1 protein, which is the most paradigmatic copper-thionein, as an experimental model. Most significantly, native Cd-Cup1 complexes were purified and fully spectroscopically and spectrometrically characterized from the 301N mutant yeast strain, which allows Cup1 synthesis even in the absence of copper. These results undoubtedly revealed the presence of a Cd-S2--Cup1 species in native preparations, which were only recovered when carefully avoiding the use of ion-exchange chromatography in the purification protocol. Furthermore, complete analysis of recombinant (Escherichia coli) Zn-Cup1, Cd-Cup1, and Cu-Cup1 and those complexes that result from Zn/Cd and Zn/Cu replacements in vitro and acidification/renaturalization processes yielded a comprehensive and comparative overview of the metal-binding abilities of Cup1. Overall, we consider the main conclusions of this study to go beyond the mere study of the particular Cup1 MT, so that they should be considered to delineate a new point of view on the interaction between copper-thioneins and divalent metal ions, still an unexplored aspect in MT research. Definitive evidence that sulfide ligands are also constituents of native metal-metallothionein (MT) complexes (see picture) is provided. Therefore, this investigation goes beyond the study of a particular MT and complements previous results about the presence of sulfide moieties in recombinant MT samples. A new point of view on the interaction between copper thioneins and divalent metal ions is delineated. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
KW - Bioinorganic chemistry
KW - Circular dichroism
KW - Metalloproteins
KW - Metallothioneins
KW - Sulfide ligands
UR - https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=uab_pure&SrcAuth=WosAPI&KeyUT=WOS:000284061400014&DestLinkType=FullRecord&DestApp=WOS_CPL
U2 - 10.1002/chem.201001125
DO - 10.1002/chem.201001125
M3 - Article
C2 - 20839184
SN - 0947-6539
VL - 16
SP - 12363
EP - 12372
JO - Chemistry - A European Journal
JF - Chemistry - A European Journal
IS - 41
ER -