Thermally triggered metal binding by recombinant Thermus thermophilus manganese superoxide dismutase, expressed as the apo-enzyme.
Manganese superoxide dismutase from the extremely thermophilic eubacterium Thermus thermophilus has been cloned and expressed at high levels in a mesophilic host (Escherichia coli) as a soluble tetrameric protein mainly present as the metal-free apo-enzyme. Incubation of the purified apo-enzyme with manganese salts at ambient temperature did not restore superoxide dismutase activity, but reactivation could be achieved by heating the protein with Mn(II) at higher temperatures, approaching the physiological growth temperature for T. thermophilus. Heat annealing followed by incubation with manganese at lower temperature fails to reactivate the enzyme, demonstrating that a simple misfolding of the protein is not responsible for the observed behavior. The in vitro metal uptake is nonspecific, and manganese, iron, and vanadium all bind, but only manganese restores catalytic activity. Bound metal ions do not exchange during heat treatment, indicating that the formation of the metal complex is effectively irreversible under these conditions. The metallation process is strongly temperature-dependent, suggesting that substantial activation barriers to metal uptake at ambient temperature are overcome by a thermal transition in the apo-protein structure. A mechanism for SOD metallation is proposed, focusing on interactions at the domain interface.
Whittaker
M M
MM
Department of Biochemistry, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006, USA.
Whittaker
J W
JW
D001051:Apoenzymes / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002845:Chromatography; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*; D013997:Time Factors; D014639:Vanadium / Q000378:metabolism
D001051:Apoenzymes; D011994:Recombinant Proteins; D014639:Vanadium; D008345:Manganese; D013482:Superoxide Dismutase