Iron-binding process in the amino- and carboxyl-terminal lobes of ovotransferrin: quantitative studies utilizing single Fe3+-binding mutants.
Iron-liganding-residue mutants of ovotransferrin, Y191F and Y524F, were investigated for their Fe(3+)-binding properties. The absorption spectrum and urea gel electrophoresis verified the single iron binding on the C- and N-lobes for Y191F and Y524F, respectively. A newly developed competitive Fe(3+)-binding analysis, in which equimolar Y191F and Y524F are mixed with less Fe(3+) than saturation, enabled us to quantitatively determine the lobe preference for initial iron entry as the ratio (alpha value) of N-lobe over C-lobe. The alpha value estimated on the basis of a kinetic model was highly dependent on pH; within a pH range from 6.5 to 9.0, alpha was increased from 2 to 5 on lowering pH with an apparent sigmoid curve. On differential scanning calorimetry, single thermal transition was observed around 61 degrees C for the apo forms of Y191F, Y524F, and wild-type ovotransferrin. The Fe(3+)-loaded mutants, however, showed dual transitions at 62.4 and 82.1 degrees C in Y191F and 66.4 and 76.0 degrees C in Y524F. According to the DeltaG(AB) value that is defined as the free energy change in a target lobe induced by the iron binding on the counter lobe, marked stabilization effects by interlobe interactions were found to be induced during the major iron-binding process: upon the primary N-lobe iron binding in the iron-free C-lobe (DeltaG(AB), -2.25 kcal/mol) and upon the secondary C-lobe iron binding in the monoferric N-lobe (DeltaG(AB), -6.45 kcal/mol).
Division of Applied Life Sciences, The Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Ikuko
I
Okamoto
Kimihiko
K
Mizutani
Masaaki
M
Hirose
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002645:Chickens; D003207:Conalbumin / Q000737:chemistry* / Q000235:genetics; D005290:Ferric Compounds / Q000737:chemistry* / Q000378:metabolism; D033862:Iron-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010649:Phenylalanine / Q000235:genetics; D010843:Pichia / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011489:Protein Denaturation; D013816:Thermodynamics; D014443:Tyrosine / Q000235:genetics
D005290:Ferric Compounds; D033862:Iron-Binding Proteins; D008024:Ligands; D010446:Peptide Fragments; D003207:Conalbumin; D014443:Tyrosine; D010649:Phenylalanine