Abstract
Metal binding to the iron storage protein apoferritin is the first step in the process by which iron accumulates within the protein shell. In the present study, the stoichiometry of metal binding to apoferritin in solution has been examined using the probe ions Mn(II), VO(IV), and Cd(II) in conjunction with EPR spectroscopic and cadmium ion selective electrode measurements. Binding studies were carried out with the individual ions, in competition with one another, and in competition with Fe(II), Fe(III), and Tb(III). All three probe ions show binding stoichiometries near 0.3 and 0.7 metal ion per subunit, close to the theoretically predicted values of 0.33 and 0.67 for the binding of one and two metal ions, respectively, per three subunits. These results in conjunction with other data are consistent with the binding of one, and possibly two, metal ions within each of the eight hydrophilic channels which are located on 3-fold axes leading to the interior of the protein. Pairs of cadmium binding sites have been located in these channels by x-ray crystallography (Rice, D. W., Ford, G. C., White, J. L., Smith, J. M. A., and Harrison, P. M. (1983) Adv. Inorg. Biochem. 5, 39-49). The possibility that some metal binding occurs elsewhere on the protein is not precluded by the present data, however. In competition experiments between various metal ions, approximately 0.3 metal ion per subunit is readily displaced implying common binding sites in the channels for all of them. The stoichiometry of Mn(II) displacement by Fe(II) is less clear. Oxidation of Fe(II) to Fe(III) by molecular oxygen in the presence of Mn(II) regenerates some Mn(II) binding on the protein, suggesting migration of iron(III) to other protein sites, or perhaps to core.
Highlights
From theTDepartment of Chemistry, University of New Hampshire, Durham, New Hampshire 03824 and the $Department of Chemistry, East Tennessee State University, Johnson City, Tennessee37604
Metal binding to the iron storagperotein apoferritin is the firststep in theprocess by which iron accumulates withinthe protein shell
The stoichiometry of metal binding to apoferritin in solution has been examined using the probe ions Mn(II), VO(IV), and Cd(I1) in conjunction with EPR spectroscopic and cadmium ion selective electrode measurements
Summary
Of Mn(I1) concentrations at both pH 6.2 (Fig. 2, inset) and pH 7.0 (not shown) to establish whether weaker binding . Restoration of Mn(I1) binding at pH 7 following oxidation was observed withsolutions having Fe(II)/ apoferritin subunit ratios of 0.5, 1.0, and 1.5 (Fig. 8). The measurement of[Cd2+],, is affected by high concentrations of protein, a binding study was carried out using 0.1 mM apoferritin (Fig. 11).Analysis of the data of this latter titration according to Equation 1yielded nl = 0.28 and Kl = 1.4. VO(IV) Binding Competition with Cd(II)-Competition between Cd(I1)and VO(1V) for the apoprotein binding sites was studied by following the amplitude of the vanadyl(1V) EPR spectrum as Cd(I1) wastitrated intoa solution of the VO(1V)apoferritin complex. A plot of electrode potential uersus the Tb(III)/subunit ratio as Tb(II1) was added to Cd(I1)-apoferritin showed that Tb(II1) ion readily displaced Cd(I1) until approximately 0.25 mol of Tb(III)/subunit was added, after which displacement was less pronounced (Fig. 15)
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