Anions transferrins

The coupling between iron and anion binding is a critical component of transferrin function. In the absence of anion, transferrins bind Fe " weakly and nonspecifically. The putative anion site identified crystal-lographically is in an electrostatically positive region, adjacent to the side chain of an arginine and the amino terminus of an a helix. The anion site does not bridge the two domains, and so does not function directly as a latch that closes the cleft around the iron. It is possible that the anion may partially compensate for the presence of several basic residues in the cleft that enhance iron binding. 

In vivo uptake of iron by transferrins usually involves its addition as a ferric-chelate complex, to prevent hydrolytic attack on the ferric ion (211). Complexes such as ferric citrate and ferric nitrilotriacetate are commonly used. Under these conditions, kinetic schemes for the uptake of iron by transferrins have identified five steps in the formation of the specific metal-anion-transferrin ternary complex (120). These may be summarized as follows. 

The determination of the structure of the iron transporter, ferric-binding, protein (hFBP)t from Haemophilus influenzae (Bruns et ah, 1997) at 0.16 nm resolution shows that it is a member of the transferrin superfamily, which includes both the transferrins and a number of periplasmic binding proteins (PBP). The PBPs transport a wide variety of nutrients, including sugars, amino acids and ions, across the periplasm from the outer to the inner (plasma) membrane in bacteria (see Chapter 3). Iron binding by transferrins (see below) requires concomitant binding of a carbonate anion, which is located at the N-terminus of a helix. This corresponds to the site at which the anions are specifically bound in the bacterial periplasmic sulfate- and... 

The iron-binding sites have been characterized by crystallographic studies on several transferrins, and in Figure 5.7 (Plate 7) that of the N-lobe of human lactoferrin is presented. The 3+ charge on the ferric ion is matched by the three anionic ligands Asp-63, Tyr-95 and Tyr-188 (the fourth, His-249, is neutral), while the charge on the carbonate anion is almost matched by the positive charge on Arg-124 and the... 

Fe(III) displacement of Al(III), Ga(III), or In(III) from their respective complexes with these tripodal ligands, have been determined. The M(III)-by-Fe(III) displacement processes are controlled by the ease of dissociation of Al(III), Ga(III), or In(III) Fe(III) may in turn be displaced from these complexes by edta (removal from the two non-equivalent sites gives rise to an appropriate kinetic pattern) (343). Kinetics and mechanism of a catalytic chloride ion effect on the dissociation of model siderophore-hydroxamate iron(III) complexes chloride and, to lesser extents, bromide and nitrate, catalyze ligand dissociation through transient coordination of the added anion to the iron (344). A catechol derivative of desferrioxamine has been found to remove iron from transferrin about 100 times faster than desferrioxamine itself it forms a significantly more stable product with Fe3+ (345). 

In addition to the amino acid side chains mentioned above, a number of other low molecular weight ligands are found in metalloproteins. These include cyanide and carbon monoxide, which we will describe later in this chapter. Here we consider carbonate and phosphate anions in the context of the super family of iron-binding proteins, the transferrins. 

Detailed pictures of the iron-binding sites in transferrins have been provided by the crystal structures of lactoferrin (Anderson et ai, 1987, 1989 Baker etai, 1987) and serum transferrin (Bailey etal., 1988). Each structure is organized into two lobes of similar structure (the amino- and carboxy-terminal lobes) that exhibit internal sequence homology. Each lobe, in turn, is organized into two domains separated by a cleft (Fig. 3 and 10). The domains have similar folding patterns of the a//3 type. One iron site is present in each lobe, which occupies equivalent positions in the interdomain cleft. The same sets of residues serve as iron ligands to the two sites two tyrosines, one histidine, and one aspartate. Additional extra density completes the octahedral coordination of the iron and presumably corresponds to an anion and/or bound water. The iron sites are buried about 10 A below the protein surface and are inaccessible to solvent. 

The molecular details of the physiologically critical step of iron release from serum transferrin are unclear. The anion may play an important role in this process by providing a handle for modulating the affinity of transferrin for iron. If the anion were to be removed (perhaps by protonation followed by dissociation), then the binding of iron to transferrin would be greatly weakened, facilitating dissociation of the metal. It seems... 

Figure for Problem 19-4. Absorption spectra lor the reaction of xylenol orange with VO2 at pH 6.0. [From D. C. Harris and M. H. Geib, Binding Xylenol Orange to Transferrin Demonstration of Metal-Anion Linkage." Biochim. Biophys. Acta I960,623,1.J... 

Milk transferrin (lactoferrin51a b c also found in leukocytes), hen egg transferrin (ovotransferrin),52 52a and rabbit and human serum transferrin54 543 all have similar structures. Each Fe3+ is bonded to oxygen anions from two tyrosine side chains, an aspartate carboxy-late, an imidazole group, and the bound carbonate ion (Fig. 16-2B). 

The significance of the two sites in transferrin has been much discussed. The sites are distinguishable spectroscopically and have different affinities for iron, which may be dependent on the anion used. The two sites release iron at different rates in a pH-dependent manner. The site in the C-terminal half of human serum transferrin (once designated the A site) retains its iron at pH 6.0 and so is the acid-stable site. The site on the N-terminal half is the acid-labile site. 

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