Abstract
Ferritins are nearly ubiquitous iron storage proteins playing a fundamental role in iron metabolism. They are composed of 24 subunits forming a spherical protein shell encompassing a central iron storage cavity. The iron storage mechanism involves the initial binding and subsequent O2-dependent oxidation of two Fe2+ ions located at sites A and B within the highly conserved dinuclear "ferroxidase center" in individual subunits. Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site (site C) located on the inner surface of the protein shell close to the ferroxidase center. We report the structures of five E. coli ferritin variants and their Fe3+ and Zn2+ (a redox-stable alternative for Fe2+) derivatives. Single carboxyl ligand replacements in sites A, B, and C gave unique effects on metal binding, which explain the observed changes in Fe2+ oxidation rates. Binding of Fe2+ at both A and B sites is clearly essential for rapid Fe2+ oxidation, and the linking of FeB2+ to FeC2+ enables the oxidation of three Fe2+ ions. The transient binding of Fe2+ at one of three newly observed Zn2+ sites may allow the oxidation of four Fe2+ by one dioxygen molecule.
Highlights
Unlike animal ferritins and the heme-containing bacterioferritins, the Escherichia coli ferritin possesses an additional iron-binding site located on the inner surface of the protein shell close to the ferroxidase center
We report the structures of five E. coli ferritin variants and their Fe3؉ and Zn2؉ derivatives
Examples include the R2 protein of ribonucleotide reductase [5], the hydroxylase component of the methane monoxygenase (MMO)1 [6], and a homodimeric protein isolated from anaerobic bacteria, which has a di-iron center in its N-terminal domain and an Fe-Cys4 cluster in its C-terminal domain [8]
Summary
Agreement of each refined model to the data, number of atoms, and waters in each model, average temperature factor for main chain atoms, and root mean square (r.m.s.) deviation from ideality for bond lengths and angles. Each subsequent model was refined with the program TNT [37] with cycles of rigid body, xyz, and xyz with B factor refinement. After convergence, (3 Fo Ϫ 2 Fc )exp(i␣c) electron density maps were calculated, and to these the individual models were rebuilt using the program FRODO [38], and the known metal binding sites [13] were inspected for the presence of metal. Additional solvent molecules were added using the program PEAKMAX [36] until all high electron density features had been explained. Iterative cycles of model building and refinement gave final models containing ϳ200 solvent molecules and overall reliability indices (R) of Ͻ20%. The geometry of each final model was checked using PROCHECK [39], and the individual refinement statistics are given in Tables I and II
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