TY - JOUR
T1 - Calometric studies on the tight binding metal interactions of Escherichia coli manganese superoxide dismutase
AU - Mizuno, Kazunori
AU - Whittaker, Mei M.
AU - Bächinger, Hans Peter
AU - Whittaker, James W.
PY - 2004/6/25
Y1 - 2004/6/25
N2 - Escherichia coli apomanganese superoxide dismutase, prepared by removing the native metal ion under denaturing conditions, exhibits thermally triggered metal uptake behavior previously observed for thermophilic and hyperthermophilic superoxide dismutases but over a lower temperature range. Differential scanning calorimetry of aposuperoxide dismutase and metalated superoxide dismutase unfolding transitions has provided quantitative estimates of the metal binding affinities for manganese superoxide dismutase. The binding constant for Mn(II) (KMn(II) = 3.2 × 108 M-1) is surprisingly low in light of the essentially irreversible metal binding characteristic of this family of proteins and indicates that metal binding and release processes are dominated by kinetic, rather than thermodynamic, constraints. The kinetic stability of the metalloprotein complex can be traced to stabilization by elements of the protein that are independent of the presence or absence of the metal ion reflected in the thermally triggered metalation characteristic of these proteins. Binding constants for Mn(III), Fe(II), and Fe(III) complexes were estimated using quasireversible values for the unfolding enthalpy and ΔCp for apo-Mn superoxide dismutase and the observed T m values for unfolding the metalated species in the absence of denaturants. For manganese and iron complexes, an oxidation state-dependent binding affinity reflects the protein perturbation of the metal redox potential.
AB - Escherichia coli apomanganese superoxide dismutase, prepared by removing the native metal ion under denaturing conditions, exhibits thermally triggered metal uptake behavior previously observed for thermophilic and hyperthermophilic superoxide dismutases but over a lower temperature range. Differential scanning calorimetry of aposuperoxide dismutase and metalated superoxide dismutase unfolding transitions has provided quantitative estimates of the metal binding affinities for manganese superoxide dismutase. The binding constant for Mn(II) (KMn(II) = 3.2 × 108 M-1) is surprisingly low in light of the essentially irreversible metal binding characteristic of this family of proteins and indicates that metal binding and release processes are dominated by kinetic, rather than thermodynamic, constraints. The kinetic stability of the metalloprotein complex can be traced to stabilization by elements of the protein that are independent of the presence or absence of the metal ion reflected in the thermally triggered metalation characteristic of these proteins. Binding constants for Mn(III), Fe(II), and Fe(III) complexes were estimated using quasireversible values for the unfolding enthalpy and ΔCp for apo-Mn superoxide dismutase and the observed T m values for unfolding the metalated species in the absence of denaturants. For manganese and iron complexes, an oxidation state-dependent binding affinity reflects the protein perturbation of the metal redox potential.
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U2 - 10.1074/jbc.M400813200
DO - 10.1074/jbc.M400813200
M3 - Article
C2 - 15082717
AN - SCOPUS:3042602322
SN - 0021-9258
VL - 279
SP - 27339
EP - 27344
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -