The unique kinetics of iron release from transferrin: the role of receptor, lobe-lobe interactions, and salt at endosomal pH

Abstract

Transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal. Human serum transferrin (hTF) carries one iron in each of two similar lobes. Understanding the detailed mechanism of iron release from each lobe of hTF during receptor-mediated endocytosis has been extremely challenging because of the active participation of the transferrin receptor (TFR), salt, a chelator, lobe-lobe interactions, and the low pH within the endosome. Our use of authentic monoferric hTF (unable to bind iron in one lobe) or diferric hTF (with iron locked in one lobe) provided distinct kinetic end points, allowing us to bypass many of the previous difficulties. The capture and unambiguous assignment of all kinetic events associated with iron release by stopped-flow spectrofluorimetry, in the presence and in the absence of the TFR, unequivocally establish the decisive role of the TFR in promoting efficient and balanced iron release from both lobes of hTF during one endocytic cycle. For the first time, the four microscopic rate constants required to accurately describe the kinetics of iron removal are reported for hTF with and without the TFR. Specifically, at pH 5.6, the TFR enhances the rate of iron release from the C-lobe (7-fold to 11-fold) and slows the rate of iron release from the N-lobe (6-fold to 15-fold), making them more equivalent and producing an increase in the net rate of iron removal from Fe(2)hTF. Calculated cooperativity factors, in addition to plots of time-dependent species distributions in the absence and in the presence of the TFR, clearly illustrate the differences. Accurate rate constants for the pH and salt-induced conformational changes in each lobe precisely delineate how delivery of iron within the physiologically relevant time frame of 2 min might be accomplished.

Fig 1

Representative normalized stopped-flow fluorescence iron release progress curves in the presence and absence of the sTFR. A) Overlay of Fe_NhTF, Fe_ChTF and Fe₂hTF, B) Overlay of Lock_NhTF, Lock_ChTF and Fe₂hTF, C) Overlay of Fe_NhTF/sTFR, Fe_ChTF/sTFR and Fe₂hTF/sTFR, D) Overlay of Lock_NhTF/sTFR, Lock_ChTF/sTFR and Fe₂hTF/sTFR. In each experiment, one syringe contained protein (375 nM) in 300 mM KCl and the other contained iron removal buffer (200 mM MES, pH 5.6, 300 mM KCl and 8 mM EDTA). Samples were excited at 280 nm and emission was monitored using a 320 nm cut-on filter; the temperature was set at 25°C.

Fig. 2

Pathways of iron release ± sTFR. A) Iron release pathways of Fe₂hTF in the absence of the sTFR, B) and the presence of sTFR. Primary pathways taken are indicated by solid black arrows, alternative pathway indicated by dashed black arrows. The specific construct to isolate the rate constants are indicated below the arrows. Rate constants ± errors at the 95% confidence level from multiple kinetic runs were obtained from curve fitting of the data to models described in the text and in Supplemental Data and are indicated for each construct. In Fig. 2B, k_1N (= 2.8 min^-1) and k_2C (= 7.2 min^-1) were held fixed at the values for Lock_ChTF and Fe_ChTF, respectively, during fitting of the Fe₂hTF/sTFR data (See Supplemental Data).

Fig. 3

Species-time distributions in the absence and in the presence of the sTFR. Distribution curves were calculated using equations 1 – 4 (Table 1) with the following rate constants: A) k_1N = 17.7 min^-1, k_1C = 0.72 min^-1, k_2N = 24.8 min^-1 and k_2C = 0.65 min^-1 and B) k_1N = 2.8 min^-1, k_1C = 5.5 min^-1, k_2N =1.4 min^-1 and k_2C = 7.2 min^-1.

Fig. 4

Salt effect on iron release from various hTF constructs in the absence and presence of the sTFR. A) Only the rate constant for iron release from the N-lobe at the various salt concentrations (indicated in the legend in Fig. 4B) is shown for Fe₂hTF, Fe_NhTF, Lock_ChTF and the isolated N-lobe at pH 5.6. Except for the salt concentration, the conditions are exactly as indicated in the legend to Fig. 1. B) Both k₁ and k₂ as a function of salt concentration are presented for the two monoferric hTF species bound to the sTFR under the same conditions as above.

Scheme I