title: The displacement of copper by iron at the specific binding sites of ovotransferrin.
abstract: We have examined the kinetics and mechanism by which iron can displace copper at the specific metal-binding sites of ovotransferrin. Fe2+ was added to Cu2+-ovotransferrin-CO3(2-) in the presence of NaHCO3 and ambient O2. The reaction has been followed by standard and stopped-flow spectrophotometry, EPR spectroscopy and analysis of chromogen-reactive Fe2+. The reaction is best described as triphasic. An initial jump in absorbance takes place in the first 2 s. In the next minute there is a further increase in absorbance and shift in the spectral maximum from 440 to 446 nm. The third phase is complex. The bulk of the spectrophotometric change, a decrease in absorbance with a shift to a maximum of 453 nm, lasts approx. 3 min. Minor spectral and EPR changes, however, take place over the next several hours. Chromogenic analysis of Fe2+ indicates that approx. 1 min is required to oxidize the Fe2+. EPR spectra reveal the formation of an Fe3+-ovotransferrin complex within the first 20 s; however, this lacks the characteristic doublet of specific Fe3+-ovotransferrin-CO3(2-). The simultaneous presence of specific Cu2+-ovotransferrin-CO3(2-) and Fe3+-ovotransferrin-CO3(2-) signals suggests a period in which the protein specifically binds both metal ions perhaps resulting from a differential reactivity of the two metal-binding sites. The addition of Cu(NO3)2 to Fe3+-ovotransferrin-CO3(2-) resulted in a complex with specific Fe3+ and non-specific Cu2+. The EPR spectrum of this complex and the final product of our displacement reaction were virtually identical. Distinct parallels in reaction of Cu2+-ovotransferrin-CO3(2-) with Fe(NH4)2(SO4)2, Fe(NO3)3 and Fe3+-nitrilotriacetic acid were observed. A reaction sequence involving the binding and oxidation of non-specific Fe2+ followed by Cu2+ displacement by Fe3+ at the specific sites and binding of non-specific Cu2+ is suggested.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843.
authors.forename: X X
authors.initials: XX
authors.lastname: Chen
authors.forename: N
authors.initials: N
authors.lastname: Fas
authors.forename: G W
authors.initials: GW
authors.lastname: Bates
mesh_terms: D000818:Animals; D001665:Binding Sites; D003207:Conalbumin / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D004527:Egg Proteins / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds; D005297:Ferrozine / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D009566:Nitrates; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D013053:Spectrophotometry
chemical_list: D004527:Egg Proteins; D005290:Ferric Compounds; D009566:Nitrates; D003207:Conalbumin; D005297:Ferrozine; D003300:Copper; D007501:Iron; D009571:Nitrilotriacetic Acid; C025302:ferric nitrate; C020326:ferric nitrilotriacetate