Iron-Binding Site of Transferrin

Typically only 30% of the iron binding sites of transferrin are occupied. Thus it retains sufficient excess binding capacity to ensure that it is never completely... 

Figure 19. A model for the anion- and iron-binding sites of transferrin depicted assuming an interlocking-site hypothesis. The protein furnishes five ligands to the metal in the iron binding site three tyrosines and two histidines. The carbonate ion binds to an arginine in the anion-binding site and functions as a sixth ligand to the metal center. The carbonate forms a bridge between the metal- and the anion-binding sites in the active center (36).

Figure 3 Examples of metal cofactors in proteins (a) the zinc center of carbonic anhydrase, (b) the blue-copper center of plastocyanin, (c) the iron center in 2,3-dihydroxybiphenil dioxygenase, (d) the iron binding site of transferrin, and (e) the dinuclear copper site of Cu/ in cytochrome c oxidase.

Because normally only about one third of the iron binding sites of transferrin are occupied by Fe ", serum transferrin has considerable reserve iron binding capacity. This is called the serum unsaturated iron binding capacity. The TIBC is a measurement of the maximum concentration of iron that transferrin can bind. The serum TIBC varies in disorders of iron metabolism. It is often increased in iron deficiency and decreased in chronic inflammatory disorders or malignancies, and it is often decreased also in hemochromatosis. 

H.G. van Eijk, W.L. van Noort, M.J. Kroos and C. van der Heul, Analysis of the iron-binding sites of transferrin by isoelectric focussing, J. Clin, Chem. Clin. Biochem., 16, 557-560 (1978). 

Figure 8.14 Structures of alternative conformations of the iron-binding sites of the recombinant N-lobe of human transferrin. (Reprinted with permission from MacGillivray et al., 1998. Copyright (1998) American Chemical Society.)...

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. 

Early experimental evidence in support of the hypothesis that an attack on the anion is at the heart of the iron-exchange mechanism (53) was soon corroborated by work from several laboratories (54, 55, 88). Replacing carbonate with oxalate at the specific anion-binding site of transferrin results in a relatively stable ternary Fe(III)-transferrin-oxalate complex. Over the time course of many hours or even days the oxalate complex slowly reverts to the physiologic Fe(III)-transferrin-carbonate form, but since in vitro studies seldom require more than an hour or two, the biologic properties of the oxalate complex can be tested. 

The serum unsaturated iron binding capacity and total iron binding capacity (TIBC) are determined by addition of sufficient Fe to saturate iron binding sites on transferrin. The excess Fe " is removed (e.g., by adsorption with light magnesium carbonate [MgCOs] powder), and the assay for iron content is then repeated. From this second measure-... 

Binding those metal ions in a metalloprotein usually prevents them from entering into these types of reactions. For example, transferrin, the iron-transport enzyme in serum, is normally only 30 percent saturated with iron. Under conditions of increasing iron overload, the empty iron-binding sites on transferrin are observed to fill, and symptoms of iron poisoning are not observed in vivo until after transferrin has been totally saturated with iron. Ceruloplasmin and metallothionein may play a similar role in preventing copper toxicity. It is very likely that both iron and copper toxicity are largely due to catalysis of oxidation reactions by those metal ions. 

Figure 4 The iron-binding site of the transferrin N-lobe showing hydrogen bonds to the carbonate ion. The iron atom is shown as a small green sphere and for clarity bonds invoiving the iron atom are not shown. Figure created using pdb coordinates IJnf...

Figure 11 The iron-binding site of recombinant N-lobe human transferrin expressed in Pichia pastoris. Five oxygen (red) atoms and one nitrogen (blue) coordinate the iron (yellow). The oxygen donors are Tyr95, Tyrl88, Asp 63, and carbonate. The nitrogen donor is His249. The coordinates were obtained from the...

Leibman, A., and Aisen, P. (1979) Distribution of iron between the binding sites of transferrin in serum methods and results in normal human subjects. Blood 53 1058-1065. 

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... 

A recently obtained high resolution structure of two crystal forms of the N-lobe of human serum transferrin (at 0.16 and 0.18 nm resolution) shows disorder at the iron-binding sites (MacGillivray et ah, 1998). Model building and refinement show... 

It seems clear that complexes 28 and 29 both enter cancer cells by transferrin-mediation. Tumor cells are known to have a high density of transferrin receptors, and this provides a route for the uptake of ruthenium (175). In normal blood plasma, transferrin is only one-third saturated with Fe(III) and therefore vacant sites are available for Ru(III) binding. Baker et al. have shown by X-ray crystallography that complex 29 binds to His-253 of apolactoferrin, one of the Fe(III) ligands in the iron binding cleft of the N-lobe, with displacement of a chloride ligand (176). Ruthenium(III) is well known to have a high affinity for solvent-exposed His side chains of proteins (177). Complex... 

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