Inequivalent Contribution of the Five Tryptophan Residues in the C-Lobe of Human Serum Transferrin to the Fluorescence Increase when Iron is Released

Abstract

Human serum transferrin (hTF), with two Fe3+ binding lobes transports iron into cells. Diferric hTF preferentially binds to a specific receptor (TFR) on the surface of cells and the complex undergoes clathrin dependent receptor-mediated endocytosis. The clathrin-coated vesicle fuses with an endosome where the pH is lowered, facilitating iron release from hTF. On a biologically relevant timescale (2-3 min), the factors critical to iron release include pH, anions, a chelator and the interaction of hTF with the TFR. Previous work, in which the increase in the intrinsic fluorescence signal was used to monitor iron release from the hTF/TFR complex, established that the TFR significantly enhances the rate of iron release from the C-lobe of hTF. In the current study, the role of the five C-lobe Trp residues in reporting the fluorescence change has been evaluated (± sTFR). Only four of the five recombinant Trp→ Phe mutants produced well. A single slow rate constant for iron release is found for the monoferric C-lobe (FeC hTF) and the four Trp mutants in the FeC hTF background. The three Trp residues equivalent to those in the N-lobe differed from the N-lobe and each other in their contributions to the fluorescent signal. Two rate constants are observed for the FeC hTF control and the four Trp mutants in complex with the TFR: kobsC1 reports conformational change(s) in the C-lobe initiated by the TFR and kobsC2 is ascribed to iron release. Excitation at 295 nm (Trp only) and at 280 nm (Trp and Tyr) reveals interesting and significant differences in the rate constants for the complex.

How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH

“…Iron release from the C lobe of hTF in the absence of the TFR is extremely slow and unaffected by the N lobe (18). The C lobe features a triad of residues (Lys534-Arg632-Asp634) that appears to control the rate constant of iron release in the absence of the TFR (31,32). Iron release from the C lobe in the presence of the TFR proceeds by a different mechanism and is 7-to 11-fold faster than in the absence of the TFR (19).…”

The long history of iron in the Universe and in health and disease

“…Thus when iron is released the fluorescent signal increases and the absorbance decreases. In a testament to the power of recombinant expression in combination with mutagenesis (see below), the contribution of each of the well-distributed individual tryptophan residues (3 in the N-lobe and 5 in the C-lobe) has been determined by making single and double point mutants [311,312]. Recent work provides evidence that, in addition to Forster resonance energy transfer, static quenching contributes to the change in the fluorescent signal when iron is released from the N-lobe [311].…”

Kinetics of iron release from transferrin bound to the transferrin receptor at endosomal pH

“…Although the kinetics of iron release from hTF at pH 5.6 are relatively slow in comparison to most enzymatic processes, many techniques monitoring iron release rates (PEG precipitation, absorbance studies and steady-state Trp fluorescence) were unable to measure early kinetic events in the process. This challenge has recently been overcome by the use of stopped-flow absorbance and fluorescence [51, 52, 62]. The use of the stopped-flow fluorescence method to measure the kinetics of iron release not only captures rapid kinetic events, but is also very sensitive (requiring only nM concentrations of protein) and is reproducible [62].…”

“…Therefore, a significant increase in the intrinsic tryptophan fluorescence of hTF is observed when iron is removed from hTF: iron removal from Fe 2 hTF, Fe N hTF and Fe C hTF results in a 368%, 74% and 71% increase the fluorescence intensity, respectively [50]. Mutagenesis studies on both the N- and C-lobes of hTF have clearly determined which Trp residues (3 in the N-lobe and 5 in the C-lobe) contribute to the observed increase in fluorescence upon iron removal [51, 52]. Moreover, the utilization of Trp analogues established that the increase in the fluorescent signal that is observed when iron is removed from hTF/TFR complexes is probably attributable to the Trp residues in hTF and not the 22 Trp residues of the TFR homodimer (note that these experiments were carried out using the soluble portion of the TFR, designated sTFR) [53].…”