M�ssbauer studies of electrophoretically purified monoferric and diferric human transferrin

Abstract

Electrophoretically purified 57Fe-enriched monoferric and diferric human transferrins and selectively labeled complexes ([C-56Fe,N-57Fe]transferrin and [C-57Fe,N-56Fe]transferrin) were studied by Mössbauer spectroscopy. The data were recorded at 4.2 K over a wide range of applied magnetic fields (0.05-6 T) and were analyzed by a spin-Hamiltonian formalism. Characteristic hyperfine parameters were found and the obtained zero-field splitting parameters (D = 0.25 +/- 0.05 cm-1 and E/D = 0.30 +/- 0.02) agree with previous electron paramagnetic resonance (EPR) findings. The weak-field spectra of the [N-57Fe]transferrin are slightly broader than those of the [C-57Fe]transferrin, indicating that the N-terminal iron site may be more heterogeneous. However, the absorption line positions and the relative intensities of the subspectra originating from the three Kramers doublets of each Fe3+ site are identical. Thus the electronic structures of the two iron sites can be described by the same set of spin-Hamiltonian parameters, indicating that the ligand environments for the two sites are the same, as suggested by the recent X-ray crystallographic studies. This suggestion is further supported by the observation that the strong-field spectra of the two monoferric transferrins are indistinguishable. The selectively labeled mixed-isotope transferrins exhibit spectra that are identical to those of the corresponding monoferric 57Fe-enriched transferrins, implying that the occupation of one iron site has little or no effect on the immediate environment of the other site, a finding that is not surprising since the two sites are separated by approximately 4.2 nm.

The role of transferrin in the mechanism of cellular iron uptake

“…Identical results have been obtained with rabbit serum transferrin and human lactoferrin [15,16]. Recent M6ssbauer studies have also confirmed the similarity between the two ironbinding sites in human transferrin [17].…”

Disorder at metal sites in proteins: A high-frequency-EMR study

“…Analysis of the sharp features at g = 4.3 in the X-band spectra, together with spectra at Qband (35 GHz), give an estimate of the value of the zero-field splitting parameter, D, of about 0.25 cm -' for this sharp component [14]. Mossbauer studies lead to a similar conclusion [15]. Details of the EMR spectra vary with changes in salt or glycerol in the solvent, and substantial changes are seen when the synergistic anion is changed from carbonate to oxalate or other bidentate chelators [22].…”

“…Recently, the relaxation times of ferric transferrin complexes were measured directly and the results indicated, that the relaxation-determined line widths were 1 MHz or less in the range 5 to 30 K, confirming that the apparent shapes are not relaxation-determined [13]. The zerofield splitting of ferric iron in transferrin is on the order of the X-band quantum (0.3 cm -') [14,15], so substantial differences in shape and effective gvalues are expected between X-and W-band spectra. Lastly, catalase was chosen as an example of a heme protein containing high-spin ferric iron.…”

Simulation Methods for Looping Transitions

“…The approximation that results from substituting Eqs. [12] and [15] The cubic approximation for ϭ 29.4°, 29.7°, 29.9°, and 29.975°is plotted in Fig. 4 as an unbroken curve; it passes through each of the "exact" points at the resolution of the figure in this paper.…”

“…The zero-field splitting of magnetic energy levels for ferric transferrin (D ϳ 0.25 cm Ϫ1 ) (14,15) is on the order of the X-band quantum (v/c ϳ 0.3 cm…”