Table of Contents
ISRN Biophysics
Volume 2012 (2012), Article ID 124803, 5 pages
Research Article

Density Functional Theory Study on Metal-Binding Energies for Human Serum Transferrin-Metal Complexes

1Faculty of Nutritional Sciences, The University of Morioka, Takizawa, Iwate 020-0183, Japan
2Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1–3 Kagurazaka, Shinjyuku-ku, Tokyo 162-8601, Japan

Received 16 December 2011; Accepted 3 January 2012

Academic Editors: D. Bulone and P. G. Fajer

Copyright © 2012 Tetsuya Sakajiri et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The absolute values of the metal-binding energies of human serum transferrin (Tf) N-lobe, | Δ E | , were calculated using the density functional theory and were found to increase in magnitude in the following order: Fe(III)>Ga(III)>Al(III)>Cu(II)>Zn(II)>Ni(II). The calculated energies were well correlated with the logarithmic values of the reported metal-binding constants of Tf, which had been experimentally determined, with a correlation coefficient of 0.96. In the estimation of the binding energies, the solvation energies (solvent effect) of free metal ions were a very important factor. The results provide a theoretical explanation for the binding of Fe(III) to Tf, which produces sufficient energy to induce a conformational transition of the Tf molecule, making it possible to interact with Tf receptor 1.

1. Introduction

Serum transferrin (Tf), the iron-transport protein in vertebrates, is a glycosylated single polypeptide with a molecular mass of approximately 80 kDa [1]. It consists of two major lobes, N- and C-lobe, which have about 40% homology between their amino acid sequences [2]. Each lobe is further subdivided into two similarly sized domains (N1 and N2; C1 and C2) and possesses a single iron-binding site in the interdomain cleft (N- and C-site) [2]. Iron bound at each site is octahedrally coordinated by six ligands: two tyrosine residues, one aspartic acid residue, one histidine residue, and a synergistic anion (a bidentate carbonate ion in a physiological condition), of which one oxygen atom is in interaction with η NH of arginine residue [3, 4].

In addition to ferric ions, the iron-binding sites are capable of binding various other metal ions [5, 6]. Among these, ferric ions most tightly bind to Tf, giving a binding constant of 1020~23 M−1 [5, 6]. Diferric Tf (Fe(III)2Tf) binds to Tf receptor 1 (TfR1) at the cell surface (pH 7.4) [79]. The binding of the Fe(III)2Tf complex to TfR1 is followed by endocytosis and iron release in the acidic environment (pH 5.6) of the endosome. The iron-free Tf (apoTf) remains bound to the TfR1 in the endosome. This adduct returns to the cell surface, and the apoTf is dissociated from the TfR1 at pH 7.4, which allows the recycling of both the proteins [7].

Many studies have been carried out on the physicochemical properties of Tf-metal complexes. In particular, regarding the stability of a Tf-metal complex, the metal ion radius has been thought to be one of the most important factors [10]. For instance, in the lanthanum series, wherein the elements have very similar chemical properties, the binding stabilities of the metal ions are correlated with their radii, that is, a Tf complex with a lanthanoid ion with a large radius is more unstable than one with a small radius [10]. However, for Fe(III), which is a key metal ion, although the radius of Fe(III) (0.64 Å) is larger than that of Al(III) (0.54 Å) [6], the Tf-Fe(III) complex is much more stable than the Tf-Al(III) complex (binding constant: 1013~14 M−1 [6]). Thus, the metal ion radius alone is not necessarily the dominant factor in the stability of a Tf-metal complex.

The binding of Fe(III) to Tf induces a great conformation change that interacts strongly with TfR1 [1113], that is, upon Fe(III)-loading, the N1 and N2, and C1 and C2 domains undergo conformational changes, which bring about a transition of the cleft from the open form of apoTf to the closed form of Fe(III)2Tf.

In human circulation, Tf is saturated with iron to only approximately 30% [14]. This suggests that there is a possibility of binding various metal ions to Tf in the circulation. Cobalt (III), gallium (III), and bismuth (III) tightly bind to Tf with metal-binding constants of 10~22 M−1 [15], 1019~20 M−1 [5, 6], and 10~19 M−1 [6], respectively. The Tf complexes of these metal ions interact with TfR1 at pH 7.4, but the binding affinities of Co(III)2Tf, Ga(III)2Tf, and Bi(III)2Tf to TfR1 are approximately 11-, 500-, and 2000-fold lower than that of Fe(III)2Tf, respectively [11]. Al(III)2Tf, which has a binding constant of 1013~14 M−1 [6], does not interact with TfR1 [11, 16]. Recent studies [12, 13] by our group showed that the structure of Al(III)2Tf has an intermediate form between the closed and open forms of the Tf domains, resulting in the absence of the binding of Al(III)2Tf to TfR1. Thus, the Tf/TfR1-mediated uptake of a biologically toxic element such as Al by cells is prevented. On the other hand, radioisotopes such as 67Ga(III) [17] and 60Co(III) [18] are able to be used as medical diagnostic and/or therapeutic regents by forming stable complexes with Tf for use against tumor cells, which contain an abundance of TfR1.

Tf complexes of metal ions having high affinities to Tf can bind to TfR1. The binding energy of a metal ion to Tf is thought to contribute to the conformational change in the Tf molecule. Therefore, it is physiologically important to understand the factors responsible for the formation of the Tf-metal complex.

In order to analyze these factors, we calculated the intrinsic binding energies of several metal ions, including Fe(III), using the density functional theory (DFT) by (1) considering in vacuo conditions for the binding site and free metal ions and then (2) taking the solvent effect for the free metal ions in water into consideration. The calculated results were evaluated by investigating their correlation with the reported experimental binding constants [6]. The solvation energy of the metal ion was shown to be an important factor in its affinity to Tf.

2. Methods

The 3D coordinates of Fe(III)-binding Tf N-lobe were obtained from the X-ray data of the crystal structure of iron-binding human N-lobe Tf (PDB ID: 1A8E) [3] and used for the DFT calculation. The metal-binding ligands and their surrounding atomic groups used for the calculation were defined as follows: functional groups of the amino acid residues (Asp-63, Tyr-95, Tyr-188, His-249) and C O 3 2 , Arg-124 interacting with C O 3 2 , and two water molecules [3]. We denote the atomic groups in the metal-binding environment in total as the binding site (BS) for this calculation.

The binding energy between the BS and each metal in vacuo or with the solvation effect is defined by the following formula: Δ 𝐸 (binding energy) = 𝐸 (metal-binding BS in vacuo) − [ 𝐸 (BS in vacuo) + 𝐸 (free metal ion in vacuo or water)]. We employed Gaussian03W with the B3LYP/6-31G** basis set [19] for the ab initio DFT calculations of the complexes with Fe(III), Ga(III), Al(III), Cu(II), Zn(II), and Ni(II). The solvation energy of each free metal ion was calculated using the DFT method with a metal ion coordinated by six water molecules in the octahedral geometry as a solvation model. Furthermore, IPCM [20] with the B3LYP/6-31G** basis set was used as a reaction field model of the solvation effect for the surrounding water molecules of the solvent model at a solvent dielectric constant of 78.39.

3. Results and Discussion

First, we estimated the Fe(III)-ligand distances by geometry optimization of the Fe(III)-BS complex with an octahedral structure. Figure 1 shows the calculated bond lengths between the Fe(III) and oxygen atoms of the ligand residues, O of Asp-63, O of Tyr-95, and O of Tyr-188, as well as between a nitrogen atom of His-245 and oxygen atoms of C O 3 2 , that is, a synergistic anion. All these lengths concurred with those obtained from the X-ray crystal data (1.6 Å resolution) [3] within a deviation of only 0.1 Å (Table 1). The results of the present geometrical optimization using only the ligands directly participating in the BS as a model showed that the complex structure was not disturbed by other parts of the protein. Consequently, this metal-binding site model was evaluated to be adequate for the theoretical calculation in this study. The bond lengths between each metal ion and its ligands for the Ga(III)BS, Al(III)BS, Cu(II)BS, Zn(II)BS, and Ni(II)BS complexes are listed in Table 1. For the Zn(II)BS complex, the bond distance between Zn(II) and Tyr-188 (O) was estimated to be 7.04 Å. This may be a five-coordinate complex.

Table 1: Bond lengths between each metal ion and its ligands (calculation data and experimental data of the crystal structure).
Figure 1: Optimized structure of Fe(III)-BS complex. The unit of the bond lengths of ligand-Fe(III) is Å.

We then calculated the metal-binding energy employing the BS model defined above. This calculation was carried out for the intrinsic metal-binding energies ( Δ 𝐸 𝑖 ) under the conditions of no hydration for the free metal ions and a dielectric constant of 𝜀 = 1 (in vacuo). The calculated metal-binding energies in vacuo showed a linear relationship with the logarithmic values ( l o g 𝐾 1 ) of the reported metal-binding constants of Tf (Table 2), which had been experimentally determined [6], with a correlation coefficient of 𝛾 = 0 . 8 6 3 . As shown in Figure 2, the order of the absolute values of the calculated binding energies in vacuo ( | Δ 𝐸 𝑖 | ) with respect to the metal ions is as follows: Ga(III)>Fe(III)>Al(III)>Cu(II)>Zn(II)>Ni(II). On the other hand, the order of the experimentally obtained l o g 𝐾 1 values is as follows: Fe(III)>Ga(III)>Al(III)>Cu(II)>Zn(II)>Ni(II) [6]. The contradiction between these orders requires the calculation of metal-binding energies under conditions that are similar to the physiological ones. Therefore, we recalculated the binding energies by considering the solvation effect for free metal ions and the dielectric constant of water ( 𝜀 = 7 8 . 3 9 ). These results are also shown in Figure 2. The absolute values of calculated metal-binding energies (real metal-binding energies, Δ 𝐸 𝑟 ) showed the following order: Fe(III)>Ga(III)>Al(III)>Cu(II)>Zn(II)>Ni(II). This order was consistent with that of the experimentally obtained l o g 𝐾 1 values [6]. The correlation between them was improved, with 𝛾 = 0 . 9 5 8 (Figure 2).

Table 2: Binding energies and binding constants (experimental data).
Figure 2: Plot of the absolute value of the calculated binding energy ( | Δ 𝐸 | , kcal/mol) between each metal ion and BS versus the Experimentally obtained l o g 𝐾 1 value [6]. The open squares denote the absolute values of the binding energies in vacuo (intrinsic binding energies, | Δ 𝐸 𝑖 | ), and the correlation coefficient is 𝛾 = 0 . 8 6 3 . The filled circles denote the absolute values of the binding energies with the solvation effect (real binding energies, | Δ 𝐸 𝑟 | ) and 𝛾 = 0 . 9 5 8 .

The solvation energy of Ga(III) in solution was calculated to be −1196 kcal/mol (Table 2). The absolute value of this energy was much larger than that calculated for Fe(III), that is, | 7 8 3 | kcal/mol. During the binding of a metal ion to the BS, the metal ion has to undergo dehydration. The dehydration energy (minus solvation energy) is supplied from the metal-binding energy in vacuo ( Δ 𝐸 𝑖 ). The energy difference, that is, the intrinsic metal-binding energy − solvation energy, is the real binding energy ( Δ 𝐸 𝑟 ). The real binding energy of Fe(III) to the BS had the largest absolute value. This binding energy was consumed to induce a conformational transition to the completely closed form of Fe(III)2Tf from the open form of apoTf. Our group [13] observed the hydrodynamic radii to be 4 2 . 6 ± 0 . 1  Å for apoTf, 3 8 . 8 ± 0 . 2  Å for Al(III)2Tf, and 3 7 . 2 ± 0 . 3  Å for Fe(III)2Tf, by using dynamic light scattering. These observations support the results of the present study. Table 2 lists the values of the calculated intrinsic binding energies, solvation energies, real binding energies, and the reported experimental binding constants [6] in the l o g 𝐾 form for the metal ions used in the present study.

In conclusion, we calculated the binding energies of several metals to Tf by DFT using the model shown in Figure 1 and by considering the solvent effects for free metal ions. This provided fairly good results to theoretically explain the binding of Fe(III) to Tf, which produces sufficient energy to induce a conformational change in the Tf molecule, making it possible to interact with TfR1.


This work was supported in part by Morioka University Academic grant.


  1. J. H. Brock, Transferrins, Macmillan, London, UK, 1985.
  2. J. Wally, P. J. Halbrooks, C. Vonrhein et al., “The crystal structure of iron-free human serum transferrin provides insight into inter-lobe communication and receptor binding,” Journal of Biological Chemistry, vol. 281, no. 34, pp. 24934–24944, 2006. View at Publisher · View at Google Scholar · View at Scopus
  3. R. T. A. MacGillivray, S. A. Moore, J. Chen et al., “Two high-resolution crytal sructures of the recombinant N-lobe of human tranferrin reveal a structure change implicated in iron release,” Biochemistry, vol. 37, no. 22, pp. 7919–7928, 1998. View at Publisher · View at Google Scholar · View at Scopus
  4. D. R. Hall, J. M. Hadden, G. A. Leonard et al., “The crystal and molecular structures of diferric porcine and rabbit serum transferrins at resolutions of 2.15 and 2.60 Å, respectively,” Acta Crystallographica Section D, vol. 58, no. 1, pp. 70–80, 2002. View at Publisher · View at Google Scholar · View at Scopus
  5. W. R. Harris, Binding and Transport of Nonferrous Metals by Serum Transferrin, Springer, Berlin, Germany, 1998.
  6. H. Sun, H. Li, and P. J. Sadler, “Transferrin as a metal ion mediator,” Chemical Reviews, vol. 99, no. 9, pp. 2817–2842, 1999. View at Google Scholar · View at Scopus
  7. A. Dautry Varsat, A. Ciechanover, and H. F. Lodish, “pH and the recycling of transferrin during receptor-mediated endocytosis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 80, no. 8, pp. 2258–2262, 1983. View at Google Scholar · View at Scopus
  8. C. Harding, J. Heuser, and P. Stahl, “Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes,” Journal of Cell Biology, vol. 97, no. 2, pp. 329–339, 1983. View at Google Scholar · View at Scopus
  9. M. W. Hentze, M. U. Muckenthaler, B. Galy, and C. Camaschella, “Two to tango: regulation of mammalian iron metabolism,” Cell, vol. 142, no. 1, pp. 24–38, 2010. View at Publisher · View at Google Scholar · View at Scopus
  10. T. Yamamura, K. Ichimura, T. Tsuda et al., “Lanthanoid complex of iron-transport protein, transferrin—kinetic study on release of the metal from N and C binding sites,” Nippon Kagaku Kaishi, vol. 4, pp. 452–458, 1988. View at Google Scholar
  11. N. T. Ha-Duong, M. Hémadi, Z. Chikh, and J. M. E. H. Chahine, “Kinetics and thermodynamics of metal-loaded transferrins: transferrin receptor 1 interactions,” Biochemical Society Transactions, vol. 36, no. 6, pp. 1422–1426, 2008. View at Publisher · View at Google Scholar · View at Scopus
  12. T. Sakajiri, T. Yamamura, T. Kikuchi, and H. Yajima, “Computational structure models of apo and diferric transferrin-transferrin receptor complexes,” Protein Journal, vol. 28, no. 9-10, pp. 407–414, 2009. View at Publisher · View at Google Scholar · View at Scopus
  13. T. Sakajiri, T. Yamamura, T. Kikuchi, K. Ichimura, T. Sawada, and H. Yajima, “Absence of binding between the human transferrin receptor and the transferrin complex of biological toxic trace element, aluminum, because of an incomplete open/closed form of the complex,” Biological Trace Element Research, vol. 136, no. 3, pp. 279–286, 2010. View at Publisher · View at Google Scholar · View at Scopus
  14. J. V. Princiotto and E. J. Zapolski, “Difference between the two iron binding sites of transferrin,” Nature, vol. 255, no. 5503, pp. 87–88, 1975. View at Google Scholar · View at Scopus
  15. T. A. D. Smith, “Human serum transferrin cobalt complex: stability and cellular uptake of cobalt,” Bioorganic and Medicinal Chemistry, vol. 13, no. 14, pp. 4576–4579, 2005. View at Publisher · View at Google Scholar · View at Scopus
  16. M. Hémadi, G. Miquel, P. H. Kahn, and J. M. El Hage Chahine, “Aluminum exchange between citrate and human serum transferrin and interaction with transferrin receptor 1,” Biochemistry, vol. 42, no. 10, pp. 3120–3130, 2003. View at Publisher · View at Google Scholar · View at Scopus
  17. L. R. Bernstein, “Mechanisms of therapeutic activity for gallium,” Pharmacological Reviews, vol. 50, no. 4, pp. 665–682, 1998. View at Google Scholar · View at Scopus
  18. W. W. Shreeve, “Use of isotopes in the diagnosis of hematopoietic disorders,” Experimental Hematology, vol. 35, no. 4, pp. 173–179, 2007. View at Publisher · View at Google Scholar · View at Scopus
  19. M. J. Frisch, G. W. Trucks, H. B. Schlegel et al., Gaussian03, in: C. Revisions C.01, D.02, and E.01, Ed, Gaussian, Inc., Wallingford, 2004.
  20. J. B. Foresman, T. A. Keith, K. B. Wiberg, J. Snoonian, and M. J. Frisch, “Solvent effects—5. Influence of cavity shape, truncation of electrostatics, and electron correlation on ab initio reaction field calculations,” Journal of Physical Chemistry, vol. 100, no. 40, pp. 16098–16104, 1996. View at Google Scholar · View at Scopus