Exploring the Fe(III) binding sites of human serum transferrin with EPR at 275 GHz

We report 275 GHz EPR spectra of human serum transferrin. At this high microwave frequency the zero-field splitting between the magnetic sublevels of the high-spin Fe^3+ sites can be accurately determined. We find the zero-field splitting to be a sensitive probe of the structure of the transferrin iron-binding sites. Signals arising from iron bound to the transferrin N-lobe can clearly be distinguished from signals from iron bound to the C-lobe. Moreover, our spectra show that the structure of the iron site in the N-lobe is influenced by the presence and conformation of the C-lobe. The spectra of a series of N-lobe mutants altering the second-shell interaction of Arg124 with the synergistic anion carbonate reflect conformational changes induced at the iron site.