Conformationally gated metal uptake by apomanganese superoxide dismutase

Mei M. Whittaker, James Whittaker

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Metal uptake by apomanganese superoxide dismutase in vitro is a complex process exhibiting multiphase "gated" reaction kinetics and a striking sigmoidal temperature profile that has led to a model of conformationally gated metal binding, requiring conversion between "closed" and "open" forms. This work systematically explores the structural determinants of metal binding in both wild-type (WT) apoprotein and mutational variants as a test of mechanistic models. The pH dependence of metalation under physiological conditions (37°C) shows it is linked to ionization of a single proton with a pKa of 7.7. Size exclusion chromatography demonstrates that the apoprotein is dimeric even when it is fully converted to the open form. The role of molecular motions in metal binding has been probed by using disulfide engineering to introduce covalent constraints into the protein. While restricting motion at domain interfaces has no effect, constraining the subunit interface significantly perturbs metal uptake but does not prevent the process. Mutagenesis of residues in the active site environment results in a dramatic shift in the transition temperature by as much as 20°C or a loss of pH sensitivity. On the basis of these results, a mechanism for metal uptake by manganese superoxide dismutase involving reorientation of active site residues to form a metal entry channel is proposed.

Original languageEnglish (US)
Pages (from-to)11625-11636
Number of pages12
JournalBiochemistry
Volume47
Issue number44
DOIs
StatePublished - Nov 4 2008

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Superoxide Dismutase
Metals
Apoproteins
Catalytic Domain
Mutagenesis
Size exclusion chromatography
Transition Temperature
Reaction kinetics
Disulfides
Ionization
Gel Chromatography
Protons
Temperature
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Conformationally gated metal uptake by apomanganese superoxide dismutase. / Whittaker, Mei M.; Whittaker, James.

In: Biochemistry, Vol. 47, No. 44, 04.11.2008, p. 11625-11636.

Research output: Contribution to journalArticle

Whittaker, Mei M. ; Whittaker, James. / Conformationally gated metal uptake by apomanganese superoxide dismutase. In: Biochemistry. 2008 ; Vol. 47, No. 44. pp. 11625-11636.
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