Transferrins Nernst plots

Figure 2.3 Uncorrected (a) and corrected (b) Nernst plots for diferric (Fe2Tf), O C-terminal (FecTf) and T N-terminal (FeNTf) monoferric transferrin. Conditions [MV ] = 0.2-0.4mM [KCl] = 500 mM [MES] = 50mM at pH = 5.8 20 °C FcjTf (0.11-0.19mM in Fe) FccTf (0.18-0.22 mM in Fe) FeNTf (0.68 mM in Fe). Error bars represent standard deviations for the average of 2-3 independent experiments. Data obtained below -530 mV for Fe2Tf and FecTf in panel (a) were not used for the corrected Nernst plots in panel (b) due to the low absorbance changes measured at these low potentials. Figure adapted from ref. 6 and used with permission.

Optical spectra of transferrin C-lobe docked with the transferrin receptor showed a characteristic broad absorption band centred at 465 nm, just as in the receptor-free /zo/o-protein (Figure 2.1 inset). The intensity of this absorbance band declined as more negative potentials were applied in a spectroelectrochemistry experiment, but did not qualitatively change in its overall features. An EPR spectrum of the Fec/TfR complex at pH 5.8, recovered from the OTTLE cell after completion of spectroelectrochemical studies allowed us to conclude that the first coordination shell of Fe " in transferrin is intact and unperturbed when C-lobe is complexed with TfR. Consequently, we assume that C-lobe and Fec/TfR complex have similar if not identical Fe " and Fe binding constants, and so we take for binding of Fe " in the protein-receptor complex to be 10 M as calculated for free Tf. This value was used to correct the observed Nernst plot data by accounting for the dissociation of Fe that occurs upon reduction. Nernst plots for the observed spectroelectrochemical data for FccTf/TfR, and data corrected for Fe dissociation, are presented in Figure 2.7. The corrected plot exhibits typical Nernstian behaviour for a one-electron transfer and a E1/2 value of —285 mV. 

Figure 2.8 compares corrected Nernst plots for C-lobe half-transferrin free in solution and bound to the transferrin receptor, at endosomal pH. These data clearly demonstrate that docking iron-loaded C-lobe transferrin at the transferrin receptor at pH 5.8 makes it energetically more favourable to reduce Fe " to Fe by 200 mV. Furthermore, receptor-docking places Fe reduction in a range accessible to NADH or NADPH cofactors, consistent with the hypothesis that reduction is the initial event in iron release from transferrin in the endosome. Fe " is bound by /zTf at least 14 orders of magnitude more weakly than Fe, so that reductive release of iron bound to HTi in the transferrin-transferrin receptor complex is then physiologically and thermodynamically feasible, and the barrier to transport across the endosomal membrane is lifted. The transferrin receptor, therefore, is more than a simple conveyor of... 

Nernst plot for reduction of Fe in the C lobe of human transferrin ( , FecTf) and C-lobe transferrin complexed to the transferrin receptor (A, FecTf/TfR) at endosomal pH 5.8. Data are corrected for the dissociation of Fe. These data illustrate that redox potential of Fe in the C lobe of human transferrin is shifted positive by 200 mV when it is complexed to the transferrin receptor. Conditions Au mesh electrode [FecTf/ TfR] = 0.19mM [FecTf] = 0.20mM [MES] = 50mM at pH 5.8 [methyl viologen] = 1.4mM [KCl] = 500mM. Figure adapted from ref. 19 and used with permission. 

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