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Biochemistry 1989, 28, 9216-9221
Fe2+ Binding to Apo and Holo Mammalian Ferritin+ D. Jacobs,* G. D. Watt,*.*,$ R. B. Frankel," and G. C. Papaefthymioul Battelle Memorial Institute, Columbus, Ohio 43201, Department of Physics, California Polytechnic State University, San Luis Obispo, California 93407, and Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received March 24, 1989; Revised Manuscript Received July 3, 1989
ABSTRACT: The binding of Fe2+to both apo and holo mammalian ferritin has been investigated under anaerobic conditions as a function of pH. In the p H range 6.0-7.5, 8.0 f 0.5 Fe2+ions bind to each apoferritin molecule, but above p H 7.5, a pH-dependent Fe2+ binding profile is observed with up to 80 Fe2+ ions binding a t p H 10.0. This Fe2+ binding is reversible and is accompanied by up to two H+ being released per Fe2+ bound at p H 10.0. The Fe2+ binding to apoferritin probably occurs in the 3-fold channels. A much larger and more complex pH-dependent Fe2+ binding stoichiometry was observed for holoferritin with up to 300 Fe2+ ions binding a t p H 10.0. This pH-dependent Fe2+ binding was interpreted as Fe2+ interaction a t the FeOOH mineral surface with displacement of H+ from -OH or phosphate surface groups by the incoming Fe2+ ions. Mossbauer spectroscopic measurements using 57Fe-labeledFe2+ under anaerobic conditions showed that 57Fe2+binding to holoferritin was accompanied by electron transfer to the core, yielding 57Fe3+, presumably bound to the mineral surface. Removal of added iron by Fe2+-specific chelating agents yielded 57Fe2+,demonstrating the reversibility of this electron-transfer process. The Fe2+ bound to apo- and holoferritin is readily converted to Fe3+ by exposure to O2 and strongly retained by the respective ferritin species.
M a m m a l i a n ferritin accommodates up to 4500 iron atoms within its hollow interior in the form of an Fe"'0OH mineral core. The development of this core is generally thought [see Aisen and Listowsky (1980), Thiel (1987), and Crichton and Charloteaux-Wauters (1987) for reviews] to arise from O2 oxidation of Fe2+,resulting in Fe3+ which hydrolyzes within the protein interior. Such a sequence of events implies that Fe2+-ferritin interactions occur during core development. The interaction of Fe2+ and other metal ions with apoferritin has been studied recently (Treffry & Harrison, 1984; Chasteen & Theil, 1982; Wardeska et al., 1985; Frankel et al., 1987) in an attempt to discern and evaluate the role of Fe2+ in mammalian ferritin core formation. These studies have shown that (1) Fe2+ addition to apoferritin produces an initial complex which undergoes O2 oxidation followed by rearrangement or migration of Fe(II1) with polynuclear species ultimately forming within the ferritin core (Treffry & Harrison, 1984; Rosenberg & Chasteen, 1982; Yang et al., 1987; Bauminger et al., 1989), (2) Fe2+ initially binds presumably at or near the hydrophilic channels with a stoichiometry of 8-1 2 Fe2+/apoferritin, and a mixed-valent binuclear iron species forms when Fe2+ and the Fe(II1) core interact during early core formation (Chasteen et al., 1985; Wardeska et al., 1985, 1986), and (3) 57Fe2+binds strongly to both apo- and holoferritin, forming ferritin-bound Fe2+,in the former case, and undergoing electron exchange with the core, apparently forming core surface bound 57Fe3+in the latter case (Frankel et ai., 1987). This research was supported by the National Science Foundation Biophysics Program (Grants DMB 8512382 and 8902474) and by the Bioelectromagnetics Program of the Office of Naval Research (Contract NO0 14-88-K-0353). *Address correspondence to this author. Battelle Memorial Institute. 8 Present address: Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-021 5 . 11 California Polytechnic State University. Massachusetts Institute of Technology.
*
0006-2960/89/0428-92 16$01.50/0
Other studies have examined the properties of Fe3+-bound ferritin resulting from oxidation of Fe2+initially added at low levels to mammalian apoferritin. Mossbauer measurements (Yang et al., 1987; Bauminger et al., 1989) have demonstrated the early formation of both solitary Fe3+ and oxygen-bridged Fe(II1) species which are replaced by small Fe(II1) clusters as more iron accumulates within the ferritin core. EPR measurements (Rosenberg & Chasteen, 1982) demonstrate the presence of about 12 unique Fe3+ions that remain constant in number as the core size increases. NMR relaxometry (Koenig et al., 1986) supports the presence and constancy of six to eight unique Fe(II1) sites presumably near the outside of each ferritin molecule that are filled during early core formation and remain constant with additional iron loading. These results are all consistent with Fe2+initially binding at unique apoferritin sites with subsequent conversion to Fe3+ upon oxidation. The constant number of unique Fe3+ ions suggests a fundamental role for these species in core formation. We have examined the quantitative binding of Fe2+to both apo and holo mammalian ferritin and report that well-defined Fe2+-ferritin intermediates form in both cases. These results relate to and are discussed in terms of the early events of iron core formation in apoferritin and subsequent core development in holoferritin. MATERIALS AND METHODS Horse spleen ferritin containing 2350 and 2565 iron atoms per ferritin molecule (used for the Fe2+ binding and the Mossbauer measurements, respectively) was obtained from Sigma and passed through G-25 columns to remove loosely bound metals. Total iron content was determined by the method of Lovenberg et al. (1963). Apoferritin was prepared by the thioglycolic acid dialysis procedure (Treffry & Harrison, 1978). The concentrated apoferritin at >20 mg/mL was noticeably yellow in color but contained 6.5 indicate that Fe2+binding is complex and is probably occurring at multiple, pH-dependent binding sites (most likely deprotonated -OH or phosphate groups) present on the mineral surface. This complexity is further demonstrated by the incomplete reversibility of Fe2+ binding to holoferritin as well as the proton release results (Figure lb). Some insights into this Fez+ binding process are revealed by the Mossbauer results in Table I1 which show that Fez+ binding is accompanied by an electron-transfer reaction resulting in the formation of Fe3+. Hydrolysis of the surfacebound Fe3+ would give rise to the high proton release values and is probably responsible for the partial Fe2+ binding irreversibility. Thus, the nature of the Fe2+interactions with the surface groups on the mineral surface and how these inter-
Biochemistry, Vola28, No. 23, 1989 9221
actions promote electron-transfer and hydrolysis reactions of the bound metal ions are important questions to pursue in gaining an understanding of ferritin core development. Registry No. Fe, 1439-89-6.
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