The effect of salt and site-directed mutations on the iron(III)-binding site of human serum transferrin as probed by EPR spectroscopy.
The effects of site-directed mutation and salt on the iron(III)-binding site of the recombinant half-molecule of the N-terminal lobe (hTf/2N) of human transferrin was studied by EPR spectroscopy. Changes were observed in the EPR spectra of all variants investigated (D63S, D63C, G65R, K206Q, H207E, H249E, H249Q, K296E and K296Q) compared with that of the wild-type protein. The most pronounced changes in the metal site were caused by replacement of the coordinating residues, Asp-63 and His-249, and the non-coordinating residue Lys-296, which is located in the hinge region of the iron-binding cleft. The EPR spectral changes from replacement of other non-coordinating residues were more subtle, indicating small changes in Fe3+ coordination to the protein. The EPR spectrum of variant G65R suggests that it adopts two distinct conformations in solution, one in which the two domains forming the iron-binding cleft are closed and one in which they are open; in the latter instance Asp-63 is no longer coordinated to the Fe3+. Chloride-binding studies on hTf/2N, K206Q, H207E, K296Q and K296E showed similar binding isotherms, indicating that none of the hinge region residues replaced, i.e. Lys-206, His-207 or Lys-296, are the sites of chloride binding. The results show that the coordination environment of the Fe3+ is sensitive to structural changes from site-directed mutation of both remote and coordinated residues and also to chloride-binding and ionic strength effects.
Grady
J K
JK
Department of Chemistry, Parsons Hall, University of New Hampshire, Durham 03824-3598, USA.
Mason
A B
AB
Woodworth
R C
RC
Chasteen
N D
ND
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D012965:Sodium Chloride; D014168:Transferrin / Q000235:genetics / Q000378:metabolism*
D005290:Ferric Compounds; D014168:Transferrin; D012965:Sodium Chloride