Anion exchange in human serum transferrin N-lobe: a model study with variant His249Ala

Abstract

The removal of Fe(III) from human serum transferrin by chelators is thought to proceed through intermediate species in which the chelator becomes associated with the metal center of the protein. The visible spectral shifts associated with the formation of such intermediates in the wild-type (WT) protein are too small for reliable kinetic data to be obtained. Therefore, studies were undertaken with the recombinant N-terminal lobe variant H249A, a variant showing more pronounced spectral changes. The kinetics of the synergistic anion-exchange reaction between nitrilotriacetate (NTA) and carbonate in variant H249A was studied by stopped-flow spectrophotometry as a model for this process in the WT protein. Anion exchange occurs by two pathways at pH 7.4 and 25 degrees C: an NTA-independent dissociative pathway to form a carbonate-free intermediate Fe-H249A (Eq. 1) that subsequently reacts with NTA (Eq. 2):and an NTA-dependent associative pathway (the major pathway) in which a quaternary Fe-H249A-(CO(3))(NTA) intermediate is formed (Eq. 3), which then decays to product (Eq. 4):The reverse reaction, where HCO(3)(-) exchanges for NTA, likewise follows these two pathways. The overall apparent equilibrium constant for formation of Fe-H249A-NTA from Fe-H249A-CO(3) is K'=442 at pH 7.4. The NTA complex is favored over the carbonate complex both kinetically and thermodynamically in the pH range 7.4-8.2.