Synthetic iron chelating agents as probes of the iron coordination site and metal ion exchange kinetics of transferrin and lactoferrin

Abstract Transferrin, the serum glycoprotein of molecular weight 81,000 which transports iron in human serum, has two similar, but non-equivalent, binding sites for high-spin ferric ion. Coordination of a metal ion requires (bi-)carbonate or a functional analogue as a synergistic anion. One of the major questions regarding the binding of ferric and other metal ions by transferrin has concerned the number of tyrosine phenolate groups coordinated to the metal and whether this changes from one metal to another. We have shown, using different ultraviolet spectroscopy of metal ion binding of transferrin and the model ligand EHPG [ethylene-bis(o-hydroxyphenylglycine)], that for all metal ions there are two tyrosine phenolate groups coordinated per ion (see Fig. 1). For ions above a certain critical size, such as Pr(III), coordination of the metal becomes incomplete as only one of the binding sites is less able to accommodate such large ions. In the case of Fe(III) we have proposed that the third proton which is released upon the binding of ferric ion by transferrin is due to a hydrolysis of a coordinated water molecule. The results of continuing experiments in this area and studies for transferrin and its close relative lactoferrin will be described. Circulating transferrin in normal human beings has only about one-third of its iron binding sites occupied by Fe(III). Thus transferrin acts as an iron buffer for serium, maintaining a chemical activity of free ferric ion that corresponds to 10−24M [Fe3+]. This very large thermodynamic stability of the transferrin iron complex makes it difficult for even the most powerful chelating agents to remove iron. Furthermore, the kinetics of iron release are extremely slow even for compounds with a much greater thermodynamic stability. Thus desferrioxamine B (DFO, Fig. 2), which has a stability about 106 times that of transferrin at millimolar concentrations and pH 7.4, removes iron from transferrin with a half life of several hours. However, the tricatecholate iron-chelating agent enterobactin (Fig. 2) or its synthetic analogues 3,4-LICAMS or MECAM (Fig. 3) take up iron from transferrin at rates approximately 200 times as fast as DFO. For iron(III) removal by catecholate ligands, saturation kinetics are observed at higher ligand concentrations. The rate expression for iron removal from the protein, where L represents the tricatechol ligand and Tf represents the (bi-)carbonate complex of apotransferrin, gives the following rate expression: d[FeL] dt = kobs[Fe(transferrin-bound)] (1) where kobs = k a [L] (1 + k b [L]) (2) This rate law has been explained by the following mechanism: The application of theusual steady approximations for the intermediates given above results in the following relationships between the experimental rate constants and those in the proposed mechanism; ka = k3k2k1/[k−1(k−3 + k3)] (6) kb = k2/k−1 (7) The experimental values of these constants for the ligand 3,4-LICAMS at 0.2 mM concentration are presented in the table below for transferrin at 25 and 37°C and for lactoferrin at 37 °C. If we make the assumption that the rate constant k3 is much larger than the rate constant k−2, the ratio of ka/kb = k1. In other words, the ratio of these two experimental rate constants gives the rate for the first step of the reaction, which is assigned as a conformation change of the protein. In this conformation change the protein goes from a ‘closed’ stable form of the iron complex in which the iron center is buried 10 to 15 A below the surface of the protein and hence inaccessible either to complexing or reducing agents, to an ‘open’ form which is the stable one for the apoprotein and in which the iron binding center is near the surface of the protein and hence accessible. It is this conformation change which becomes rate determining at high ligand concentrations for extraordinarily powerful iron chelating agents. The half life for this putative conformational change at 25 °C is 22 minutes, which decrease to 8.4 minutes at 37 °C. This corresponds to an enthalpy of activation of 14 kcal per mole for the iron centers. In contrast, the half life for this process at 37 °C for lactoferrin is 1100 minutes! This extremely slow rate of the conformation change of lactoferrin may by itself explain the approximately two order of magnitude greater stability it displays toward iron compared to transferrin. Other coordination properties and relative iron transfer kinetics of these two proteins will be discussed. t001 . pM Values of Selected Fe(III) Sequestering Agents. Ligand pM a (−log[Fe3+aq]) Enterobactin 35.5 HBED b 31.0 MECAM 29.4 MECAMS 29.1 3,4-LICAMS 28.5 Me3MECAMS 26.6 Ferrioxamine B 26.6 EHPG c 26.4 TRIMCAMS 25.1 NAcMECAMS 25.0 DTPA d 24.7 Transferrin 23.6 EDTA e 22.2 Tiron f 19.5 a Calculated for 10 M ligand, 1 M Fe3+, pH 7.4. b N,N-bis(2-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid. c Ethylene-1,2-bis(2-hydroxyphenylglycine). d Diethylenetriaminepentaacetic acid. e Ethylenediaminetetraacetic acid. f 1,2-Dihydroxy-3,5-disulfobenzene.