Calorimetric studies on the tight binding metal interactions of Escherichia coli manganese superoxide dismutase.
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) (K(Mn(II)) = 3.2 x 10(8) 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 DeltaC(p) 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.
Mizuno
Kazunori
K
Department of Research, Shriners Hospital for Children, Portland, Oregon 97239, USA.
Whittaker
Mei M
MM
Bächinger
Hans Peter
HP
Whittaker
James W
JW
D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli / Q000201:enzymology*; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism*; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008345:Manganese / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011489:Protein Denaturation; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D001059:Apoproteins; D005296:Ferrous Compounds; D008345:Manganese; D013482:Superoxide Dismutase; D019791:Guanidine