Iron uptake by bovine lactoferrin from nitrilotriacetatoFe(III) [FeN(Ac)3] in the presence of bicarbonate has been investigated at pH 7.1-8.7. Deprotonated apolactoferrin interacting with bicarbonate or carbonate extracts iron from nitrilotriacetatoFe(III); the direct second-order rate constant k1 = (4.90 +/- 0.20)x10(4) M(-1) s(-1), a reverse second-order rate constant k(-1) = (1.80+/-0.05)x10(5) M(-1) s(-1), and the iron-exchange equilibrium constant K1 = 0.25+/-0.05. The newly formed iron-protein complex loses a single proton with proton dissociation constant K3a = (17+/-0.5) nM, then undergoes a modification in its conformation followed by the loss of two or three protons; the first-order rate constant k2 = (1.0+/-0.10) s(-1). This induces a new modification in the conformation; the first-order rate constant k3 = (8.75+/-0.40)x10(-3) s(-1). 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; K5a = 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.