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. Fe 2+ was added to Cu 2+-ovotransferrin-CO 3 2− in the presence of NaHCO 3 and ambient O 2. The reaction has been followed by standard and stopped-flow spectrophotometry, EPR spectroscopy and analysis of chromogen-reactive Fe 2+. 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 Fe 2+ indicates that approx. 1 min is reqired to oxidize the Fe 2+. EPR spectra reveal the formation of an Fe 3+-ovotransferrin complex within the first 20 s; however, this lacks the characteristic doublet of specific Fe 3+-ovotransferrin-CO 3 2−. The simultaneous presence of specific Cu 2+-ovotransferrin-CO 3 2− and Fe 3+-ovotransferrin-CO 3 2− s 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(NO 3) 2 to Fe 3+-ovotransferrin-CO 3 2− resulted in a complex with specific Fe 3+ and non-specific Cu 2+. The EPR spectrum of this complex and the final product of our displacement reaction were virtually identical. Distinct parallels in reaction of Cu 2+-ovotransferrin-CO 3 2− with Fe(NH 4) 2(SO 4) 2, Fe(NO 3) 3 and Fe 3+-nitrolotriacetic acid were observed. A reaction sequence involving the binding and oxidation of non-specific Fe 2+ followed by Cu 2+ displacement by Fe 3+ at the specific sites and binding of non-specific Cu 2+ is suggested.