Crucial role of intralobe peptide-peptide interactions in the uptake and release of iron by ovotransferrin

Hirofumi Kurokawa, Bunzo Mikami, Masaaki Hirose

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

Abstract

The mechanism for reversible iron binding in the N-terminal lobe of ovotransferrin was investigated by a protein fragmentation approach. The iron-saturated N-terminal half-molecule of ovotransferrin was proteolyzed into large 30-kDa (1-279) and small 6-kDa (280-332) fragments by a single cleavage with Achromobacter protease I, producing a stable nicked form. For the separation of the two fragments, denaturing conditions were required. The isolated large fragment contained all four iron-coordinating ligands and the anion-binding ligand, and according to spectroscopic titration analysis, it showed iron binding capacity. The iron-bound large fragment, however, showed a blue-shifted visible absorption spectrum and a much decreased iron stability compared with those of the intact half-molecule. Analyses by ion- exchange chromatography revealed that the large fragment reassociates with the small fragment. In order for reassociation to occur, iron must be bound to the large fragment; upon reassociation, the large fragment regained its stable iron binding capacity as well as a visible absorption spectrum almost indistinguishable from those of the intact half-molecule. These data are consistent with a two-step sequential pathway for reversible iron binding in the N-terminal lobe of ovotransferrin that includes an initial iron binding intermediate having a perturbed metal environment and its transformation into the stable iron-bound holoform by peptide-peptide interactions between the large coordinating and small noncoordinating segments.

Original languageEnglish
Pages (from-to)6671-6676
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number9
Publication statusPublished - 1994 Mar 4
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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