Transferrins, the mechanism of iron release by ovotransferrin

Abstract

Iron release from ovotransferrin in acidic media (3 , pH , 6) occurs in at least six kinetic steps. The first is a very fast (# 5 ms) decarbonation of the iron-loaded protein. Iron release from both sites of the protein is controlled by what appear to be slow proton transfers. The N-site loses its iron first in two steps, the first occurring in the tenth of a second range with second order rate constant k 1 = (2.30^0.10) Â 10 4 m 21´s21 , first order rate constant k 21 = (1.40^0.10) s 21 and equilibrium constant K 1a = (60^6) mm. The second step occurs in the second range with a second order rate constant k 2 = (5.2^0.15) Â 10 3 m 21´s21 , first order rate constant k 22 = (0.2^0.02) s 21 and equilibrium constant K 2a = (39^5) mm. Iron is afterward lost from the C-site of the protein by two different pathways, one in the presence of a strong Fe(III) ligand such as citrate and the other in the presence of weak ligands such as formate or acetate. The first step, common to both paths, is a slow proton uptake which occurs in the tens of second range with a second order rate constant k 3 = (1.22^0.03) Â 10 3 m 21´s21 and equilibrium constant K 3a = (1.0^0.1) mm. In the presence of citrate, this step is followed by formation of an intermediate complex with monoferric ovotransferrin; stability constant K LC = (0.435^0.015) mm. This last step is rate-controlled by slow proton gain which occurs in the hundred second range with a second order rate constant k 4 = (1.05^0.05) Â 10 4 m 21´s21 , first order rate constant k 24 = (1.0^0.1) Â 10 22 s 21 and equilibrium constant K 4a = (0.95^0.15) mm. In the presence of a weak iron(III) ligand such as acetate or formate, formation of an intermediate complex is not detected and iron release is controlled by two final slow proton uptakes. The first occurs in the hundred to thousand second range, second order rate constant k 5 = (6.90^0.30) Â 10 6 m 21´s21 . The last step occurs in the thousand second range. Iron release by ovotransferrin is similar but not identical to that of serum-transferrin. It is slower and occurs at lower pH values. However, as seen for serum-transferrin, it seems to involve the protonation of the amino acid sidechains involved in iron co-ordination and perhaps those implicated in interdomain H-bonds. The observed proton transfers are, then, probably controlled by the change in conformation of the binding lobes from closed when iron-loaded to open in the apo-form.Keywords: transferrin; ovotransferrin; iron metabolism; stopped-flow.Transferrins constitute the most important iron regulation system in vertebrates and some invertebrates, such as worms and insects [1]. Soluble transferrins, such as serum-transferrin, lactoferrin and ovotransferrin, are glycoproteins consisting of a single chain of about 700 amino acids and 80 kDa. These proteins have very similar 3D structures and are all bilobal. Each lobe contains an iron complexation cleft, in which the metal is co-ordinated to the side-chains of four amino acid ligands and a synergistic carbonate or bicarbonate...

The Crystal Structure of Iron-free Human Serum Transferrin Provides Insight into Inter-lobe Communication and Receptor Binding

“…Interactions between Lobes-A cooperative effect of one lobe on the other lobe has been convincingly documented in a number of studies using a variety of techniques, which include NMR of hTF (58), absorption spectra of oTF (59), pH-dependent iron release of LTF (60), calorimetric studies of both hTF and oTF (61-63), electrospray ionization mass spectrometry studies of hTF (43), iron release from monoferric LTF (64), analysis by urea gels (22), and chemical relaxation studies (65)(66)(67). Significantly, a number of these studies specifically attribute cooperative effects between the two lobes of hTF to participation of helix 12 from the C-lobe (2,68).…”

Ovotransferrin: Structure, bioactivities, and preparation

“…The rate-limiting step is the conformational change induced by the rapid removal of bound iron. It was recently reported that the protonation of iron-binding residues of ovotransferrin at acidic pH in the absence of anionic chelators is slow (Abdallah & Chahine, 1999). Studies suggested the release of iron ion from ferric transferrin requires the presence of a simple anion such as pyrophosphate, sulfate, or chloride; however, the rate of anion-mediated iron release is much slower for the C-lobe than for the N-lobe (Mizutani, Muralidhara, et al, 2001;Muralidhara & Hirose, 2000), as there are two anion binding sites in the N-lobe but only one anion-binding site in the C-lobe.…”

“…The iron binding ability confers antimicrobial activity by rendering iron unavailable for microbial growth (Abdallah & Chahine, 1999;Mason & Macgillivray, 2002). Transferrins can be divided into four types according to their occurrence in nature as shown in Table 1 (Mason & Macgillivray, 2002): (1) serum transferring (pI 7.4) is found in blood plasma where its main function is iron transfer; (2) lactoferrin (pI 8.8) is found in milk and other mammalian secretions; it binds iron more tightly over a larger pH range than other transferrin proteins and has a diverse range of biological activities including innate defense;…”

Tyrosine Hydrogen Bond Properties for the Two Binding Sites of Apoovotransferrin

“…The changes of the fluorescence spectra of apotransferrins have been analyzed by addition of transition metal, lanthanide ions or aluminum(III). [8][9][10][11][12] There is a significant difference in either metal ion binding 12,13 or removal of metal ions 14,15 for N-and Cterminal binding sites of apoovotransferrin. Whether it relates the tyrosine hydrogen bonds in solution structure of two binding sites is far from clear.…”