Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe: a structurally identified SO4(2-) binding site and its implications for the kinetic pathway.
The differential properties of anion-mediated Fe(3+) release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe(3+) release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe(3+) with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe(3+) release model in which the holo form is converted depending on the anion concentration into a "mixed ligand" intermediate that releases Fe(3+). The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 A resolution demonstrated the existence of a single bound SO(4)(2-) in the interdomain cleft, which interacts directly with Thr(461)-OG1, Tyr(431)-OH, and His(592)-NE2 and indirectly with Tyr(524)-OH. The latter three groups are Fe(3+)-coordinating ligands, strongly suggesting the facilitated Fe(3+) release upon the anion occupation at this site. The SO(4)(2-) binding structure supported the single-pathway kinetic model.
Mizutani
K
K
Research Institute for Food Science, Kyoto University, Uji, Kyoto 611 0011, Japan.
Muralidhara
B K
BK
Yamashita
H
H
Tabata
S
S
Mikami
B
B
Hirose
M
M
D000838:Anions; D001665:Binding Sites; D003207:Conalbumin / Q000737:chemistry / Q000378:metabolism*; D005290:Ferric Compounds / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D013431:Sulfates / Q000378:metabolism*
D000838:Anions; D005290:Ferric Compounds; D013431:Sulfates; D003207:Conalbumin