In-transferrin

ACS Symposium Series American Chemical Society Washington, IXJ, 1980. 

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Smith, C.A., Anderson, B.F., Baker, H.M., and Baker, E.N. 1992. Metal substitution in transferrins the crystal structure of human copper-lactoferrin at 2.1-A resolution. Biochemistry 31 4527 -533. 

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. 

Mutations that lead to amino acid changes in transferrin may alter the charge of the protein, thereby leading to shifts in the IEF pattern, which mostly resemble to either... 

Step 1 Reduce Fe3+ in transferrin to Fe2+, which is released from the protein. Commonly employed reducing agents are hydroxylamine hydrochloride (NH3OH+Cl )> thioglycolic acid, or ascorbic acid. 

The transferrins belong to the iron-tyrosinate group of proteins discussed in Section 62.1.5.5.2. Charge transfer from phenolate ligands to Fem accounts for the salmon-pink colour of transferrin. The detailed coordination environment of the iron in transferrin is not known with certainty, as... 

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. 

Fiala (45) and Fiala and Burk (46) early postulated, by analogy from the visible absorption spectra of iron transferrin and the iron complex of aspergillic acid, that iron was bound in transferrins through a hydroxamic acid-CC>2 complex. This formulation is shown in Fig. 15. Fraenkel-Conrat (48), however, could find no evidence for hydroxylamido groups in chicken ovotransferrin. He also prepared and studied the properties of several hydroxylamido proteins by the chemical introduction of the hydroxylamido groups, and found that their properties were quite different from those of the transferrins. 

Since malonate (Structure I) is able to fulfill the role of the anion in transferrin, it seemed reasonable to see whether spin-labeled derivatives of malonate could serve as probes of the active sites. Two such spin-labled derivatives were prepared and tentatively identified as having structures II (N-4-(2,2,6,6-tetramethylpiperidin-l-oxyl)malonamide) and III (N-4-(2,2,6,6-tetramethylpiperidin-l-oxyl)malonate). Similar results were obtained with each (Figure 3). Upon mixing Fe(III), transferrin, and II at low pH, and then raising the pH to near-neutrality with C02-free ammonia, the characteristic orange-red color of the ternary Fe-transferrin-anion complex is promptly displayed. However, the anticipated EPR signal of the nitroxide spin-label is not observed, presumably because it is broadened beyond detectability by its proximity... 

The studies presented in this section suggest that vanadium-48 introduced into the circulation of rats finds its way into serum transferrin and ultimately into cells as V02+. The vanadium in the cytosoi of rat liver cells is mostly found in transferrin and ferritin. Whether or not this is the normal physiological pathway for this metal has not been established by these experiments. The doses are administered over a short period of time and, in some cases, exceed by one or two orders of magnitude the concentration of V that... 

The concentrations of iron as simple solvated ions, Fe2+aq or Fe3+aq, are maintained at extremely low levels because of their damaging ability in the presence of oxygen and hydrogen peroxide. In transferrin, an important iron scavenger, the iron is well-protected and is not involved in redox chemistry. Also, in the major storage proteins, ferritin and haemosiderin, the iron is present in crystalline material inside the protein shell, and is well protected from reaction. 

A number of consequences flow from the structural duplication in transferrins. First, the degree of similarity between the two binding sites assumes considerable importance, both for biological function and for interpreting chemical and spectroscopic studies. Second, it means that stable half-molecules, each with a single binding site, can be prepared, either by limited proteolysis or by recombinant DNA methods. 

Fig. 3. Domain organization of transferrins. The N-terminal lobe (above) is divided into domains N1 and N2, and the C-terminal lobe (below), into domains Cl and C2. The two lobes are related by a screw axis, a rotation of -180°, and a translation of 25 A. The two iron sites are identified with closed circles. The connecting peptide that joins the two lobes is helical in lactoferrin (solid line) and less regular in transferrin (dashed line).

Fig. 6. Pattern of disulfide bridges commonly found in transferrins. Numbering corresponds to that introduced by Williams (87) and used in Table V. Figure taken from Bailey et al. (68), with permission.

Spectroscopic studies have consistently demonstrated the existence of multiple conformational states for the metal sites in transferrins, especially when using metal ions other than Fe3+ and anions other than C032. The differences are not necessarily related to intrinsic geometrical differences between the two sites in each molecule, but also reflect changes dependent on pH, the nature of the synergistic anion, or salt effects. 

The two sites (in transferrin, at least) also show differences in iron loading behaviour. In vitro, when Fe3+ is added as a chelate complex, there are differences in which site is preferentially loaded, depending on the nature of the chelate ligand these differences are apparently kinetically determined and differ from one transferrin to another (17). 

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