Crucial role of intralobe peptide-peptide interactions in the uptake and release of iron by ovotransferrin.
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.
Kurokawa
H
H
Research Institute for Food Science, Kyoto University, Japan.
Mikami
B
B
Hirose
M
M
D000421:Alcaligenes / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases; D002645:Chickens; D002942:Circular Dichroism; D003207:Conalbumin / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D005260:Female; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation
D010446:Peptide Fragments; D003207:Conalbumin; D007501:Iron; D002268:Carboxypeptidases; D010450:Endopeptidases