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Coordination chemistry of metallothionein, the isolated metallothionein domains and the metallothionein from N. crassa
186 Abstracts
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Hg-Cys.,- PEPTIDE COMPLEXES AS MODELS FOR METALLOPROTEINS. A. Schaffer, K. Willimann a. H. Willner, Universitat Zurich, 8057 Zurich, Switzerland. The stepwise metal binding of metallothionein (MT), a small protein of M,=6500, binding 7 metal ions with all 20 Cys residues in its native state, is believed to include the initial formation of isolated metal-Cys, complexes followed by the formation of two separated metal clusters [l]. Here we report the preparation of distinct Hg-S, species with the dodecapeptide N-Ac-P-C-O-C-P-Q-CO-C-R-R-V (O=ornithine). The ready polymerization usually observed for sulfur complexes with small inorganic ligands was circumvented by cyclization of the peptide (Orn-carboxyterminus). Thus, a monomeric complex (Hg:peptide = 1:l) was isolated while with the linear derivative a monomeric and a Hg-linked dimeric structure is formed depending on the metal to peptide ratio. A significant red shift of the lowest energy absorption band (280 + 305 nm) is manifested by their UV, CD and MCD spectra due to the transformation of 56% of the terminal thiolates in monomers to bridging sulfurs in dimeric complexes. With these data a cluster formation mechanism for MT may be deduced. [l] J.H.R. Kagi and A. Schaffer, Biochemistry 27,8509 (1988)
COORDINATION CHEMISTRY OF METALLOTHIONEIN, THE ISOLATED METALLOTHIONEIN DOMAINS AND THE METALLOTHIONEIN FROM N. cra.r~a . Timothv E. Eleren, Michael F. Reed, F. Jon Kull, Kristen G. Dillon, Thomas L. Ciardelli and Dean E. Wilcox*, Department of Chemistry, Dartmouth College, Hanover, NH 03755, U.S.A. Vibrational spectroscopic methods have been used to study the coordination chemistry of metallothionein (MT) and to probe the structure of MT reconstituted with different metal ions (Zn(II), Cd@), TcO(III), Cu(I) and Ni(II)). Vibrational bands have been observed by Raman spectroscopy in the 250 - 400 cm-l region and have been assigned as terminal and bridging metal-sulfur stretching modes. A unique low frequency band appears at 138 cm-l in the spectra of all metal (M) loaded MT and has been assigned as a S-M-S bending mode; this feature appears to be characteristic of Cys-Metal-Cys binding. Our studies have now been extended to the isolated MT domains and MT from N. crassa which we have successfully prepared using solid phase peptide synthesis. This methodology is now being used to modify the primary structure of the domains to investigate the contributions of individual residues to the thermodynamics and structure of metal ion binding to metallothionein.
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LOW-TEMPERATURE MCD AND EPR STUDIES OF FE(II)METALLOTHIONEIN. M. T. Werth and M. K. Johnson, University of Georgia, Athens, GA 30602, USA. The binding of Fe(R) by apometallothionein has been investigated by electron paramagnetic circular dichroism and low-temperature magnetic In agreement with previous results (1) rabbit liver resonance spectroscopies. metallothionein was found to bind a maximum of 7 Fe(I1) ions, with cluster The formation occurring when more than 4 Fe(I1) ions are bound at pH 8.5. results indicate that all the iron in fully-loaded Fe(I1) metallothionein is accommodated in trinuclear and tetranuclear Fe(II)-thiolate clusters with S = 2 and S = 0 ground states, respectively. The spectroscopic studies reveal heterogeneity in both mononuclear and trinuclear centers exhibiting S = 2 ground states. [l] Good, M. and Vasak, M., Biochemistry, 21, 8353 (1986).
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Hg-Cys.,- PEPTIDE COMPLEXES AS MODELS FOR METALLOPROTEINS. A. Schaffer, K. Willimann a. H. Willner, Universitat Zurich, 8057 Zurich, Switzerland. The stepwise metal binding of metallothionein (MT), a small protein of M,=6500, binding 7 metal ions with all 20 Cys residues in its native state, is believed to include the initial formation of isolated metal-Cys, complexes followed by the formation of two separated metal clusters [l]. Here we report the preparation of distinct Hg-S, species with the dodecapeptide N-Ac-P-C-O-C-P-Q-CO-C-R-R-V (O=ornithine). The ready polymerization usually observed for sulfur complexes with small inorganic ligands was circumvented by cyclization of the peptide (Orn-carboxyterminus). Thus, a monomeric complex (Hg:peptide = 1:l) was isolated while with the linear derivative a monomeric and a Hg-linked dimeric structure is formed depending on the metal to peptide ratio. A significant red shift of the lowest energy absorption band (280 + 305 nm) is manifested by their UV, CD and MCD spectra due to the transformation of 56% of the terminal thiolates in monomers to bridging sulfurs in dimeric complexes. With these data a cluster formation mechanism for MT may be deduced. [l] J.H.R. Kagi and A. Schaffer, Biochemistry 27,8509 (1988)
COORDINATION CHEMISTRY OF METALLOTHIONEIN, THE ISOLATED METALLOTHIONEIN DOMAINS AND THE METALLOTHIONEIN FROM N. cra.r~a . Timothv E. Eleren, Michael F. Reed, F. Jon Kull, Kristen G. Dillon, Thomas L. Ciardelli and Dean E. Wilcox*, Department of Chemistry, Dartmouth College, Hanover, NH 03755, U.S.A. Vibrational spectroscopic methods have been used to study the coordination chemistry of metallothionein (MT) and to probe the structure of MT reconstituted with different metal ions (Zn(II), Cd@), TcO(III), Cu(I) and Ni(II)). Vibrational bands have been observed by Raman spectroscopy in the 250 - 400 cm-l region and have been assigned as terminal and bridging metal-sulfur stretching modes. A unique low frequency band appears at 138 cm-l in the spectra of all metal (M) loaded MT and has been assigned as a S-M-S bending mode; this feature appears to be characteristic of Cys-Metal-Cys binding. Our studies have now been extended to the isolated MT domains and MT from N. crassa which we have successfully prepared using solid phase peptide synthesis. This methodology is now being used to modify the primary structure of the domains to investigate the contributions of individual residues to the thermodynamics and structure of metal ion binding to metallothionein.
Do07
DO08
LOW-TEMPERATURE MCD AND EPR STUDIES OF FE(II)METALLOTHIONEIN. M. T. Werth and M. K. Johnson, University of Georgia, Athens, GA 30602, USA. The binding of Fe(R) by apometallothionein has been investigated by electron paramagnetic circular dichroism and low-temperature magnetic In agreement with previous results (1) rabbit liver resonance spectroscopies. metallothionein was found to bind a maximum of 7 Fe(I1) ions, with cluster The formation occurring when more than 4 Fe(I1) ions are bound at pH 8.5. results indicate that all the iron in fully-loaded Fe(I1) metallothionein is accommodated in trinuclear and tetranuclear Fe(II)-thiolate clusters with S = 2 and S = 0 ground states, respectively. The spectroscopic studies reveal heterogeneity in both mononuclear and trinuclear centers exhibiting S = 2 ground states. [l] Good, M. and Vasak, M., Biochemistry, 21, 8353 (1986).