Transferrins

Abstract

Iron uptake by bovine lactoferrin from nitrilotriacetatoFe(III) [FeN(Ac) 3 ] in the presence of bicarbonate has been investigated at pH 7.1Ϫ8.7. Deprotonated apolactoferrin interacting with bicarbonate or carbonate extracts iron from nitrilotriacetatoFe(III); the direct second-order rate constant k 1 ϭ (4.90 Ϯ0.20)ϫ10 4 M Ϫ1 s Ϫ1 , a reverse second-order rate constant k Ϫ1 ϭ (1.80Ϯ 0.05)ϫ10 5 M Ϫ1 s Ϫ1 , and the iron-exchange equilibrium constant K 1 ϭ 0.25Ϯ 0.05. The newly formed iron-protein complex loses a single proton with proton dissociation constant K 3a ϭ (17Ϯ 0.5) nM, then undergoes a modification in its conformation followed by the loss of two or three protons; the first-order rate constant k 2 ϭ (1.0Ϯ 0.10) s Ϫ1 . This induces a new modification in the conformation; the first-order rate constant k 3 ϭ (8.75 Ϯ0.40)ϫ10 Ϫ3 s Ϫ1. This second modification in conformation controls the rate of iron uptake by the N site of the protein and is followed by a single proton loss; K 5a ϭ 8.0 nM. Finally, the holoprotein or the monoferric lactoferrin in their final equilibrated states are produced by a third modification in the conformation occurring in about 9000 s. The mechanism of iron uptake by lactoferrin is very similar to that of serum transferrin with a cooperativity between the C and N sites upon iron uptake but with lower rates, higher affinities and at least one more proton loss involved. These differences may be the result of slight discrepancies in the intimate structures of binding sites for serum transferrin and lactoferrin. In order to analyse the cooperativity between these iron-binding sites, the three-dimensional position of the chain of amino acid residues separating the N and C lobes of human apo-, holo-and dicopper-lactoferrin have been compared by the recognition of the three-dimensional shape dissimilarity program. The interlobe peptides of human hololactoferrin and apolactoferrin showed only 75.5 % tridimensional similarity, indicating that iron uptake affects the three-dimensional structure of the interlobe chain.

Monitoring lactoferrin iron levels by fluorescence resonance energy transfer: a combined chemical and computational study

“…Our interpretations are also consistent with those of other authors, who have envisaged the possibility that cooperativity between the C-and N-lobes occurs via dynamic event modifications of the interlobe chain, rather than the lobes themselves [18,33].…”

“…At this point, it is interesting to note that uptake of the first iron(III) by lactoferrin has been reported to occur at the C-site, at which point the N-site becomes capable of acquiring a second iron(III) ion [33]. However, the mechanism by which the N-lobe is activated once this first iron(III) ion has been bound to the Clobe is not well known and the source of debate.…”

Transferrins, the mechanism of iron release by ovotransferrin

“…This is confirmed by an X±ray diffraction study on the N-site of serum-transferrin [14]. With serum-transferrin, the release of the metal from the N-site involves a single slow proton-transfer reaction which we ascribed to an acid-base reaction occurring on the Hist ligand (Eqn 2) and probably controlled by the change of conformation from close to open occurring upon iron loss [12,16]. This was also confirmed by X-ray diffraction [5].…”

“…Most of the`acid-base like' pK a s reported in Table 1 are close to those of the side-chains of the amino acids involved in complex formation between ovotransferrin and Fe(III) and in the interdomain H-bonds present in the C-binding cleft. We can therefore cautiously speculate that after the loss of carbonate, the first step involved in iron release form the C-site of ovotransferrin is the protonation of one of the Glu or Asp engaged in the interdomain H-bonds [Eqn (16), pK a = 4]. This allows the interaction of the competing citrate ligand with the co-ordination sites of the metal left accessible by carbonate loss.…”

“…However, in neutral media ovotransferrin does behave as the iron-scavenging lactoferrin [9,16]. This may explain the duality of ovotransferrin as an iron scavenger in neutral media and/or deliverer in acidic endosomes.…”