Release of iron from transferrin by phosphonocarboxylate and diphosphonate chelating agents

The rates at which phosphonocarboxylate and diphosphonate ligands remove iron from the serum iron transport protein transferrin at 25 degrees C and pH 7.4 have been evaluated. These ligands show a combination of saturation and first-order kinetics with respect to the free ligand concentrations. The ability of the ligands to remove iron from transferrin appears to be subject to steric restrictions that are essentially identical to those associated with the ability of a ligand to substitute for the synergistic carbonate anion. This observation supports the hypothesis that the first-order component for iron removal involves a mechanism in which the rate-limiting step is the slow substitution of the synergistic carbonate by the incoming chelating agent. Studies on monoferric transferrins indicate that phosphonocarboxylates are unusually effective at removing iron from the C-terminal site of the protein. Difference UV spectroscopy has been used to show that the phosphonocarboxylates bind strongly to apotransferrin. It is suggested that the rapid release of iron from the C-terminal site may be due to the binding of the ligand to an allosteric anion-binding site in the C-terminal lobe of the protein.     

Kinetics and mechanism of exogenous anion exchange in FeFbpA–NTA: significance of periplasmic anion lability and anion binding activity of ferric binding protein A

The bacterial transferrin ferric binding protein A (FbpA) requires an exogenous anion to facilitate iron sequestration, and subsequently to shuttle the metal across the periplasm to the cytoplasmic membrane. In the diverse conditions of the periplasm, numerous anions are known to be present. Prior in vitro experiments have demonstrated the ability of multiple anions to fulfill the synergistic iron-binding requirement, and the identity of the bound anion has been shown to modulate important physicochemical properties of iron-bound FbpA (FeFbpA). Here we address the kinetics and mechanism of anion exchange for the FeFbpA–nitrilotriacetate (NTA) assembly with several biologically relevant anions (citrate, oxalate, phosphate, and pyrophosphate), with nonphysiologic NTA serving as a representative synergistic anion/chelator. The kinetic data are consistent with an anion-exchange process that occurs in multiple steps, dependent on the identity of both the entering anion and the leaving anion. The exchange mechanism may proceed either as a direct substitution or through an intermediate FeFbpA–X* assembly based on anion (X) identity. Our kinetic results further develop an understanding of exogenous anion lability in the periplasm, as well as address the final step of the iron-free FbpA (apo-FbpA)/Fe³⁺ sequestration mechanism. Our results highlight the kinetic significance of the FbpA anion binding site, demonstrating a correlation between apo-FbpA/anion affinity and the FeFbpA rate of anion exchange, further supporting the requirement of an exogenous anion to complete tight sequestration of iron by FbpA, and developing a mechanism for anion exchange within FeFbpA that is dependent on the identity of both the entering anion and the leaving anion.     

Mutations at the histidine 249 ligand profoundly alter the spectral and iron-binding properties of human serum transferrin N-lobe

Human serum transferrin is an iron-binding and -transport protein which carries iron from the blood stream into various cells. Iron is held in two deep clefts located in the N- and C-lobes by coordinating to four amino acid ligands, Asp 63, Tyr 95, Tyr 188, and His 249 (N-lobe numbering), and to two oxygens from carbonate. We have previously reported the effect on the iron-binding properties of the N-lobe following mutation of the ligands Asp 63, Tyr 95, and Tyr 188. Here we report the profound functional changes which result from mutating His 249 to Ala, Glu, or Gln. The results are consistent with studies done in lactoferrin which showed that the histidine ligand is critical for the stability of the iron-binding site [H. Nicholson, B. F. Anderson, T. Bland, S. C. Shewry, J. W. Tweedie, and E. N. Baker (1997) Biochemistry 36, 341-346]. In the mutant H249A, the histidine ligand is disabled, resulting in a dramatic reduction in the kinetic stability of the protein toward loss of iron. The H249E mutant releases iron three times faster than wild-type protein but shows significant changes in both EPR spectra and the binding of anion. This appears to be the net effect of the metal ligand substitution from a neutral histidine residue to a negative glutamate residue and the disruption of the "dilysine trigger" [MacGillivray, R. T. A., Bewley, M. C., Smith, C. A., He, Q.-Y., Mason, A. B., Woodworth, R. C., and Baker, E. N. (2000) Biochemistry 39, 1211-1216]. In the H249Q mutant, Gln 249 appears not to directly contact the iron, given the similarity in the spectroscopic properties and the lability of iron release of this mutant to the H249A mutant. Further evidence for this idea is provided by the preference of both the H249A and H249Q mutants for nitrilotriacetate rather than carbonate in binding iron, probably because NTA is able to provide a third ligation partner. An intermediate species has been identified during the kinetic interconversion between the NTA and carbonate complexes of the H249A mutant. Thus, mutation of the His 249 residue does not abolish iron binding to the transferrin N-lobe but leads to the appearance of novel iron-binding sites of varying structure and stability.     

The synergistic anion-binding sites of human transferrin: chemical and physiological effects of site-directed mutagenesis

A defining feature of all transferrins is the absolute dependence of iron binding on the concomitant binding of a synergistic anion, normally but not necessarily carbonate. Acting as a bridging ligand between iron and protein, it completes the coordination requirements of iron to lock the essential metal in its binding site. To investigate the role of the synergistic anion in the iron-binding and iron-donating properties of human transferrin, a bilobal protein with an iron binding site in each lobe, we have selectively mutated the anion-binding threonine and arginine ligands that form an essential part of the electrostatic and hydrogen-bonding network holding the synergistic anion to the protein. Preservation of either ligand is sufficient to maintain anion binding, and therefore iron binding, in the mutated lobe. Arginine is a stronger ligand than threonine, and its loss weakens carbonate and therefore iron binding, but maintains the ability of nitrilotriacetate to serve as a carbonate surrogate. Replacement of both ligands abolishes anion binding and consequently iron binding in the affected lobe. Loss of anion binding in either lobe results in a monoferric protein binding iron in normal fashion only in the opposite lobe. Both monoferric proteins are capable of transferrin receptor-dependent binding and iron donation to K562 cells, but with diminished receptor occupancy by the protein bearing iron only in the N-lobe.     

Polynuclear iron compounds in human transferrin preparations

During commonly used saturation procedures of transferrin with iron compounds, both as ferri and ferrous, polynuclear iron compounds are easily formed, even when nitrilotriacetate (NTA) is used as a strong iron ligand. The presence of these nonspecific bound irons is demonstrated with Mossbauer spectroscopy and with electronic optical spectroscopy. But no evidence, however, has been found of two different iron binding sites. Because dialysis is not able to remove all polynuclear iron, an easy method with gel filtration has been developed that does remove the polynuclear iron. Some notes are made about the often used method, in transferrin biochemistry, of saturation determination, i.e. the quotient of the absorbances of 470 and 280 nm.     

Non-transferrin bound iron measurement is influenced by chelator concentration

Release of non-protein bound iron plays an important role in the toxicity inflicted by chemotherapy in cancer patients. Since large variations have been described for different methods measuring non-transferrin bound iron (NTBI), we aimed to obtain more accurate values. After binding to the chelator nitrilotriacetic acid disodium salt (NTA) and ultrafiltration, the NTBI can be measured spectrophotometrically by the addition of thioglycolic acid (TGA) and baptophenanthroline disulfonic acid (BPT). Results demonstrated that NTBI values increased with NTA concentration. In samples incubated with 80 mM NTA, >5-fold higher NTBI values were found compared to using 10 mM NTA. Optimal concentration of NTA was established by additions of iron to serum with known latent iron-binding capacity (LIBC). Iron addition curves showed that NTBI could be measured starting from the LIBC of the serum with optimal yield after incubation with 4 mM NTA in 5 mM Tris-HCl pH 6.5, with 3 mM TGA and 6.2 mM BPT for the colour reaction. The results showed excellent correlation with 195 samples measured also by HPLC. For the spectrophotometric method, significantly higher NTBI values were measured in patient samples with maximal iron saturation compared to patients with lower iron saturation.     

The role of the synergistic phosphate anion in iron transport by the periplasmic iron-binding protein from Haemophilus influenzae

The acquisition of iron from transferrin by Gram-negative bacterial pathogens is dependent on a periplasmic ferric-ion-binding protein, FbpA. FbpA shuttles iron from the outer membrane to an inner membrane transport complex. A bound phosphate anion completes the iron co-ordination shell of FbpA and kinetic studies demonstrate that the anion plays a critical role in iron binding and release in vitro. The present study was initiated to directly address the hypothesis that the synergistic anion is required for transport of iron in intact cells. A series of site-directed mutants in the anion-binding amino acids of the Haemophilus influenzae FbpA (Gln-58, Asn-175 and Asn-193) were prepared to provide proteins defective in binding of the phosphate anion. Crystal structures of various mutants have revealed that alteration of the C-terminal domain ligands (Asn-175 or Asn-193) but not the N-terminal domain ligand (Gln-58) abrogated binding of the phosphate anion. The mutant proteins were introduced into H. influenzae to evaluate their ability to mediate iron transport. All of the single site-directed mutants (Q58L, N175L and N193L) were capable of mediating iron acquisition from transferrin and from limiting concentrations of ferric citrate. The results suggest that the transport of iron by FbpA is not dependent on binding of phosphate in the synergistic anion-binding site.     

Quantification of non-transferrin-bound iron in the presence of unsaturated transferrin.

Non-transferrin-bound iron (NTBI) has been reported to be associated with several clinical states such as thalassemia, hemochromatosis, and in patients receiving chemotherapy. We have investigated a number of ligands as potential alternatives to nitrilotriacetic acid (NTA) to capture NTBI without chelating transferrin- or ferritin-bound iron in plasma. We have established, however, that NTA is the optimal ligand to chelate the different forms of NTBI present in sera and can be adopted for utilization in the NTBI assay. NTA (80 mM) removes all forms of NTBI, while only mobilizing a small fraction of the iron bound to both transferrin and ferritin. We have compared three different detection systems for the quantification of NTA-chelated NTBI: the established HPLC-based method, a simple colorimetric method, and a method based on inductive conductiometric plasma spectroscopy. The sensitivity and reproductibility of the colorimetric method were acceptable compared with the other two methods and would be more convenient as a routine laboratory screening assay for NTBI. However, the limitations of this method are such that it can only be utilized in situations where desferrioxamine is not used and when transferrin saturation levels are close to 100%. Only the HPLC-based method is applicable for patients receiving (desferrioxamine) chelation therapy. In some diseases such as hemochromatosis, transferrin may be incompletely saturated. In such cases, to avoid in vitro donation of iron onto the vacant sites of transferrin, sodium-tris-carbonatocobaltate(III) can be added to block the free iron binding sites on transferrin. If this step is not taken, there may be an underestimation of NTBI values.     

Kinetics of iron release from ferric binding protein (FbpA): mechanistic implications in bacterial periplasm-to-cytosol Fe3+ transport

The ferric binding protein (FbpA) transports iron across the periplasmic space of certain Gram-negative bacteria and is an important component involved in iron acquisition by pathogenic Neisseria spp. (Neisseria gonorrheae and Neisseria meningitidis). Previous work has demonstrated that the synergistic anion, required for tight Fe(3+) sequestration by FbpA, also plays a key role in inserting Fe(3+) into the FbpA binding site. Here, we investigate the iron release process from various forms of holo-FbpA, Fe(3+)FbpA-X, during the course of a chelator competition reaction using EDTA and Tiron. Fe(3+)FbpA-X represents the protein assembly complex with different synergistic anions, X = PO(4)(3)(-) and NTA. Stepwise mechanisms of Fe(3+) release are proposed on the basis of kinetic profiles of these chelator competition reactions. Fe(3+)FbpA-PO(4) and Fe(3+)FbpA-NTA react differently with EDTA and Tiron during the Fe(3+)-exchange process. EDTA replaces PO(4)(3)(-) and NTA from the first coordination shell of Fe(3+) and acts as a synergistic anion to give a spectroscopically distinguishable intermediate, Fe(3+)FbpA-EDTA, prior to pulling Fe(3+) out of the protein. Tiron, on the other hand, does not act as a synergistic anion but is a more efficient competing chelator as it removes Fe(3+) from FbpA at rate much faster than EDTA. These results reaffirm the contribution of the synergistic anion to the FbpA iron transport process as the anion, in addition to playing a facilitative role in iron binding, appears to have a "gatekeeper" role, thereby modulating the Fe(3+) release process.     

New perspectives on the structure and function of transferrins.

Structure-function relationships for transferrins are discussed in the light of recent X-ray crystal structure determinations. A common folding pattern into two lobes, each comprising two domains is adopted; this allows the tight, but reversible binding of iron. Uptake and release of iron involve substantial domain movements which open and close the binding clefts. The iron binding sites are similar and the key role of the CO3(2-) anion bound with each Fe3+ can now be understood; structural differences near the iron binding sites suggest reasons for the different binding properties of serum transferrin and lactoferrin. The glycan moieties do not appear to affect the protein structure or metal binding properties; they are not clearly seen in the X-ray analyses but have been modelled. The accommodation of different metals and anions is illustrated by the crystal structures of Cu2+ and oxalate-substituted lactoferrins; Al3+ binding is of particular interest. New results on transferrin-receptor interactions with transferrin, and melanotransferrin and an invertebrate transferrin (both of which have defective C-terminal binding sites), emphasize possible functional differences between the two lobes. The availability of site-specific mutants of both transferrin and lactoferrin now offers the opportunity to probe the structural determinants of iron binding, iron release, and receptor binding.     

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin.

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin has been studied by analyzing the distribution of iron between the chelator and the proteins as a function of both ligand concentration and transferrin saturation. The kinetics of iron removal by 3-hydroxypyridin-4-ones from both transferrins is slow; in ovotransferrin it appears to be monophasic, in contrast to that observed for serum transferrin. After 24 hours incubation at a 40:1 chelator:protein molar ratio, the percentage of iron removed from Fe(III)-ovotransferrin is 50%-60%, and is somewhat higher in the case of serum transferrin, in line with the respective affinity constants for the metal. The 3-hydroxypyridin-2-ones and the 3-hydroxypyran-4-ones, both of which have lower affinities for Fe(III), remove smaller proportions of the metal. The percentage of desaturation obtained with bidentate and hexadentate pyridinones appears to be similar for both transferrin classes at chelator:protein molar ratios from 40:1. The degree of transferrin saturation influences the extent of chelator mediated iron mobilization in the case of serum transferrin, but not of ovotransferrin. 59Fe competition studies demonstrate that bidentate pyridin-4-ones are capable of donating iron to serum apotransferrin; the relative concentrations of ligand and protein influence the distribution of iron because their effective binding constants (at pH 7.4) for Fe(III) are similar.     

Iron release from transferrin induced by mixed ligand complexes of copper(II)

Summary Copper(II) complexes CuL₁L₂ with the ligand pairs 3-phosphoglycerate (PG)/ethylenediamine (en), phosphoserine (PS)/ethylenediamine, phosphoserine/malonate (mal) are shown to be effective in inducing the release of both iron atoms from di-ferric transferrin (Fe2Tf; human serum transferrin) at pH 7.3 in 1 M NaCl at 25°C. Half-times of the reaction with Cu(PG)(en)^– were less than 1 min at 0.02 M concentration. The iron(III) products are polynuclear hydroxo complexes. There is weaker interaction with Cu(PS) ₂ ^4– and virtually none with Cu(serine)(en) nor Cu(PS)(2,2-bipyridyl)^–, revealing crucial effects of the combined ligand sphere including the phosphomonoester group. The results suggest that the release of iron from Fe2Tf, or from either monoferric transferrins, occurred due to the breakdown of the stability of iron binding in conjunction with the expulsion of the synergistic anion carbonate (or oxalate). The active copper(II) complexes are postulated to be models of membrane components that could liberate iron from transferrin succeeding its uptake at the receptor sites of cells.Abbreviations PG phosphoglycerate - PS phosphoserine - en ethylenediamine - Fe2Tf diferric transferrin - Fe_cTf and Fe_NTf transferrin with iron bound to the lobe containing the C- or N-terminus, respectively - apoTf apotransferrin - K-3 all-cis-1,3,5-tris(trimethylammonio)-2,4,6-cyclo-hexanetriol - NTA nitrilotriacetic acid; bipy, 2,2-bipyridine; mal, malonate     

Site selectivity in the binding of inorganic anions to serum transferrin

Equilibrium constants for the sequential binding of two anions at the specific metal-binding sites of apotransferrin have been measured by difference ultraviolet spectroscopy in 0.1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 7.4 and 25 degrees C. Log K1 values for phosphate, phosphite, sulfate, and arsenate fall in the narrow range of 3.5-4.0, while the log K1 for bicarbonate is 2.73. No binding is observed for nitrate, perchlorate, or borate. A dinegative charge appears to be the most important criterion for anion binding. Equilibrium constants have also been measured for binding of anions to both forms of mono(ferric)transferrin. There appears to be a very small site selectivity (0.2 to 0.4 log units) for phosphate, arsenate, and phosphite that favors binding to the N-terminal site, but there is no detectable selectivity for binding of sulfate or bicarbonate. Comparison of the binding affinities and anion selectivity with literature data on anion-binding to protonated macrocyles and cryptates strongly supports the existence of specific anion-binding sites on the protein. Binding constants were also measured in 0.01 M Hepes. The anionic sulfonate group of the buffer appears to have a small effect on anion binding.     

Mutation of the iron ligand His 249 to Glu in the N-lobe of human transferrin abolishes the dilysine "trigger" but does not significantly affect iron release

Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive "trigger" involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half-molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.     

Identification of possible kinetically significant anion-binding sites in human serum transferrin using molecular modeling strategies

Certain anions have been shown experimentally to influence the rate of iron release from human serum transferrin (HST), implying the existence of one or more allosteric kinetically significant anion-binding (KISAB) sites on or near the surface of the protein. A rank-ordered selection of potential HST KISAB sites has been obtained using a novel three-stage molecular modeling strategy. The crystal structure of HST (1A8E.pdb) was first subjected to a heuristic analysis, in which positively charged and hydrogen-bonding residues on or near the surface of the protein were identified. In this stage, a preliminary electrostatic potential map was also calculated, yielding six preliminary sites. Next, energy-grid calculations were conducted in order to identify anion-protein interaction energy minima, which resulted in the inclusion of three additional sites. Finally, three anions already shown experimentally to demonstrate varied effects on HST iron-release kinetics were placed at each potential site; molecular dynamics and molecular mechanics calculations were performed in order to elucidate the hydrogen-bonding environment around each anion of the protein as well as to calculate anion-protein-binding energies.     

Dual role of Lys206-Lys296 interaction in human transferrin N-lobe: iron-release trigger and anion-binding site.

The unique structural feature of the dilysine (Lys206-Lys296) pair in the transferrin N-lobe (hTF/2N) has been postulated to serve a special function in the release of iron from the protein. These two lysines, which are located in opposite domains, hydrogen bond to each other in the iron-containing hTF/2N at neutral pH but are far apart in the apo-form of the protein. It has been proposed that charge repulsion resulting from the protonation of the dilysines at lower pH may be the trigger to open the cleft and facilitate iron release. The fact that the dilysine pair is positively charged and resides in a location close to the metal-binding center has also led to the suggestion that the dilysine pair is an anion-binding site for chelators. The present report provides comprehensive evidence to confirm that the dilysine pair plays this dual role in modulating release of iron. When either of the lysines is mutated to glutamate or glutamine or when both are mutated to glutamate, release of iron is much slower compared to the wild-type protein. This is due to the fact that the driving force for cleft opening is absent in the mutants or is converted to a lock-like interaction (in the case of the K206E and K296E mutants). Direct titration of the apo-proteins with anions as well as anion-dependent iron release studies show that the dilysine pair is part of an active anion-binding site which exists with the Lys296-Tyr188 interaction as a core. At this site, Lys296 serves as the primary anion-binding residue and Tyr188 is the main reporter for electronic spectral change, with smaller contributions from Lys206, Tyr85, and Tyr95. In iron-loaded hTF/2N, anion binding becomes invisible as monitored by UV-vis difference spectra since the spectral reporters Tyr188 and Tyr95 are bound to iron. Our data strongly support the hypothesis that the apo-hTF/2N exists in equilibrium between the open and closed conformations, because only in the closed form is Lys296 in direct contact with Tyr188. The current findings bring together observations, ideas, and experimental data from a large number of previous studies and shed further light on the detailed mechanism of iron release from the transferrin N-lobe. In iron-containing hTF/2N, Lys296 may still function as a target to introduce an anion (or a chelator) near to the iron-binding center. When the pH is lowered, the protonation of carbonate (synergistic anion for metal binding) and then the dilysine pair form the driving force to loosen the cleft, exposing iron; the nearby anion (or chelator) then binds to the iron and releases it from the protein.     

The role of the anion-binding site of transferrin in its interaction with the reticulocyte

Specific and tight binding of Fe(III) by transferrin does not occur unless a suitable anion is concomitantly bound. Bicarbonate, which normally occupies the anion binding site of the protein, may be replaced by an oxalate ion. The resulting ternary complex of Fe(III), transferrin and oxalate is less than 35% as effective as the bicarbonate complex in serving as an iron donor for heme synthesis by the reticulocyte. However, the binding of transferrin to the reticulocyte is not altered by the substitution of oxalate for bicarbonate. When both the oxalate and bicarbonate forms are incubated with reticulocytes, the uptake of iron from the bicarbonate complex is substantially depressed. The free oxalate ion, at the same concentration as the ternary Fe-transferrin-oxalate complex, does not alter the uptake of iron by reticulocytes from the native form of transferrin. The ternary Fe-transferrin-malonate complex is also less efficient than the bicarbonate complex as an iron donor to the reticulocyte, but the effect is less striking than that observed with the oxalate complex. The hypothesis is advanced that the mechanism of iron uptake from transferrin during the transferrin-reticulocyte interaction first entails an attack upon the anion bound to the protein, following which iron release to the heme-synthesizing apparatus of the cell takes place.     

Structural insight into the novel iron‐coordination and domain interactions of transferrin‐1 from a model insect,Manduca sexta

Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly. Vertebrate transferrins coordinate iron through interactions with two tyrosines, an aspartate, a histidine, and a carbonate anion, and conformational changes that occur upon iron binding and release have been described. Much less is known about the structure and functions of insect transferrin‐1 (Tsf1), which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms. Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf1 differs from that of the vertebrate transferrins. Here we report the first crystal structure (2.05 Å resolution) of an insect transferrin. Manduca sexta (MsTsf1) in the holo form exhibits a bilobal fold similar to that of vertebrate transferrins, but its carboxyl‐lobe adopts a novel orientation and contacts with the amino‐lobe. The structure revealed coordination of a single Fe³⁺ ion in the amino‐lobe through Tyr90, Tyr204, and two carbonate anions. One carbonate anion is buried near the ferric ion and is coordinated by four residues, whereas the other carbonate anion is solvent exposed and coordinated by Asn121. Notably, these residues are highly conserved in Tsf1 orthologs. Docking analysis suggested that the solvent exposed carbonate position is capable of binding alternative anions. These findings provide a structural basis for understanding Tsf1 function in iron sequestration and transport in insects as well as insight into the similarities and differences in iron homeostasis between insects and humans.     

Magnesium potentiation of iron-transferrin binding

The binding of iron to transferrin was studied by loading iron (III) onto apotransferrin in a chloride and a nitrilotriacetate form. When magnesium was added, a marked increase occurred in both the rate of iron binding and the maximum level of iron loaded on transferrin utilizing either iron salt. In the absence of magnesium the amount of iron required to achieve 50 percent saturation of the binding sites was 1.6 x 10(-4) M, whereas when magnesium was added, only about one-third as much iron (0.54 x 10(-4) M) was required. These data suggest an allosteric effect on transferrin by magnesium which potentiates iron (III) binding.     

Evolution of the transferrin family: conservation of residues associated with iron and anion binding

The transferrin family spans both vertebrates and invertebrates. It includes serum transferrin, ovotransferrin, lactoferrin, melanotransferrin, inhibitor of carbonic anhydrase, saxiphilin, the major yolk protein in sea urchins, the crayfish protein, pacifastin, and a protein from green algae. Most (but not all) contain two domains of around 340 residues, thought to have evolved from an ancient duplication event. For serum transferrin, ovotransferrin and lactoferrin each of the duplicated lobes binds one atom of Fe (III) and one carbonate anion. With a few notable exceptions each iron atom is coordinated to four conserved amino acid residues: an aspartic acid, two tyrosines, and a histidine, while anion binding is associated with an arginine and a threonine in close proximity. These six residues in each lobe were examined for their evolutionary conservation in the homologous N- and C-lobes of 82 complete transferrin sequences from 61 different species. Of the ligands in the N-lobe, the histidine ligand shows the most variability in sequence. Also, of note, four of the twelve insect transferrins have glutamic acid substituted for aspartic acid in the N-lobe (as seen in the bacterial ferric binding proteins). In addition, there is a wide spread substitution of lysine for the anion binding arginine in the N-lobe in many organisms including all of the fish, the sea squirt and many of the unusual family members i.e., saxiphilin and the green alga protein. It is hoped that this short analysis will provide the impetus to establish the true function of some of the TF family members that clearly lack the ability to bind iron in one or both lobes and additionally clarify the evolutionary history of this important family of proteins.