title: Tertiary structural changes and iron release from human serum transferrin.
abstract: Iron release from human serum transferrin was investigated by comparison of the extent of bound iron, measured by charge transfer absorption band intensity (465 nm), with changes observed by small-angle solution X-ray scattering (SAXS) for a series of equilibrated samples between pH 5.69 and 7.77. The phosphate buffers used in this study promote iron release at relatively high pH values, with an empirical pK of 6.9 for the convolved release from the two sites. The spectral data reveal that the N-lobe release is nearly complete by pH 7.0, while the C-lobe remains primarily metal-laden. Conversely, the radius of gyration, Rg, determined from the SAXS data remains constant between pH 7.77 and 7.05, and the evolution of Rg between its value observed for the diferric protein at pH 7.77 (31.2+/-0.2 A) and that of the apo protein at pH 5.69 (33.9+/-0.4 A) exhibits an empirical pK of 6.6. While Rg is effectively constant in the pH range associated with iron release from the N-lobe, the radius of gyration of cross-section, Rc, increases from 16.9+/-0.2 A to 17.6+/-0.2 A. Model simulations suggest that two different rotations of the NII domain relative to the NI domain about a hinge deep in the iron-binding cleft of the N-lobe, one parallel with and one perpendicular to the plane of the iron-binding site, can be significantly advanced relative to their holo protein positions while yielding constant Rg and increased Rc values consistent with the scattering data. Rotation of the CII domain parallel with the C-lobe iron-binding site plane can partially account for the increased Rg values measured at low pH; however, no reasonable combined repositioning of the NII and CII domains yields the experimentally observed increase in Rg.
authors.affiliation: Biochemistry and Biotechnology Group, CST-4, MS-J586, Los Alamos National Laboratory, Los Alamos, NM, 87545, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Mecklenburg
authors.forename: R J
authors.initials: RJ
authors.lastname: Donohoe
authors.forename: G A
authors.initials: GA
authors.lastname: Olah
mesh_terms: D000818:Animals; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D007781:Lactoferrin / Q000737:chemistry; D008958:Models, Molecular; D009010:Monte Carlo Method; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D014168:Transferrin / Q000737:chemistry* / Q000378:metabolism; D014965:X-Rays
chemical_list: D014168:Transferrin; C028283:diferric transferrin; D007501:Iron; D007781:Lactoferrin