Dissociation of human copper-zinc superoxide dismutase dimers using chaotrope and reductant: Insights into the molecular basis for dimer stability

Peter A. Doucette, Lisa J. Whitson, Xiaohang Cao, Virgil Schirf, Borries Demeler, Joan Selverstone Valentine, Jeffrey C. Hansen, P. John Hart

Research output: Contribution to journalArticlepeer-review

127 Scopus citations

Abstract

The dissociation of apo- and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carbozyethyl)phosphine (TCEP) has been analysed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo- and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0-3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 → Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dinner by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.

Original languageEnglish (US)
Pages (from-to)54558-54566
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number52
DOIs
StatePublished - Dec 24 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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