TERTIARY STRUCTURAL-CHANGES AND IRON RELEASE FROM HUMAN SERUM TRANSFERRIN

Iron release from human serum transferrin was investigated by comparis on of the extent of bound iron, measured by charge transfer absorption band intensity (465 nm), with changes observed by small-angle solutio n 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 ir on release at relatively high pH values, with an empirical pK of 6.9 f or 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, R- g, determined from the SAXS data remains constant between pH 7.77 and 7.05, and the evolution of R-g between its value observed for the dife rric protein at PH 7.77 (31.2 +/- 0.2 Angstrom) and that of the apo pr otein at pH 5.69 (33.9 +/- 0.4 Angstrom) exhibits an empirical pK of 6 .6. While R-g is effectively constant in the pH range associated with iron release from the N-lobe, the radius of gyration of cross-section, R-c, increases from 16.9 +/- .0.2 Angstrom to 17.6 +/- 0.2 A. Model s imulations suggest that two different rotations of the NII domain rela tive to the NT domain about a hinge deep in the iron-binding cleft of the N-lobe, one parallel with and one perpendicular to the plane of th e iron-binding site, can be significantly advanced relative to their h ole protein positions while yielding constant R-g and increased R-c va lues consistent with the scattering data. Rotation of the CII domain p arallel with the C-lobe iron-binding site plane can partially account for the increased R-g values measured at low pH; however, no reasonabl e combined repositioning of the NII and CII domains yields the experim entally observed increase in R-g.