The displacement of copper by iron at the specific binding sites of ovotransferrin

• Published in: Biochimica et biophysica acta Vol. 992; no. 2; pp. 160 - 167
• Main Authors: Xian-Xuan, Chen, Fas, Norberto, Bates, George W.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 18.08.1989; Elsevier; North-Holland
• Subjects: analogs & derivatives; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; chicks; Conalbumin; Conalbumin - metabolism; copper; Copper - metabolism; Copper displacement; Egg Proteins; Egg Proteins - metabolism; eggs; Electron Spin Resonance Spectroscopy; Ferric Compounds; Ferrozine; Ferrozine - metabolism; Fundamental and applied biological sciences. Psychology; Iron; Iron - metabolism; Kinetics; metabolism; Metal binding site; Metalloproteins; Nitrates; Nitrilotriacetic Acid; Nitrilotriacetic Acid - analogs & derivatives; Other metalloproteins; Ovotransferrin; Proteins; Spectrophotometry; transferrin; Copper displacement; Iron; Metal binding site; Ovotransferrin; Hepes; Ferroprotein; Absorption spectrometry; EPR spectrometry; Metal complex; Binding site; Kinetics; Copper; Mechanism of action
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(89)90005-6

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.