Thermally triggered metal binding by recombinant Thermus thermophilus manganese superoxide dismutase, expressed as the apo-enzyme

Mei M. Whittaker, James Whittaker

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)34751-34757
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number49
DOIs
StatePublished - Dec 3 1999

Fingerprint

Thermus thermophilus
Superoxide Dismutase
Manganese
Metals
Temperature
Enzymes
Hot Temperature
Proteins
Vanadium
Eubacterium
Coordination Complexes
Growth temperature
Escherichia coli
Metal ions
Catalyst activity
Ion exchange
Thermodynamic properties
Heating
Iron
Salts

ASJC Scopus subject areas

  • Biochemistry

Cite this

Thermally triggered metal binding by recombinant Thermus thermophilus manganese superoxide dismutase, expressed as the apo-enzyme. / Whittaker, Mei M.; Whittaker, James.

In: Journal of Biological Chemistry, Vol. 274, No. 49, 03.12.1999, p. 34751-34757.

Research output: Contribution to journalArticle

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