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NMR reveals pathway for ferric mineral precursors to the central cavity of ferritin
Authors:Paola Turano  Daniela Lalli  Isabella C. Felli  Elizabeth C. Theil  Ivano Bertini
Affiliation:aMagnetic Resonance Center and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy; ;bCouncil on BioIron at Children’s Hospital Oakland Research Institute, 5700 Martin Luther King, Jr. Way, Oakland, CA 94609; and ;cDepartment of Nutritional Sciences and Toxicology, University of California, Berkeley, CA 94720
Abstract:Ferritin is a multimeric nanocage protein that directs the reversible biomineralization of iron. At the catalytic ferroxidase site two iron(II) ions react with dioxygen to form diferric species. In order to study the pathway of iron(III) from the ferroxidase site to the central cavity a new NMR strategy was developed to manage the investigation of a system composed of 24 monomers of 20 kDa each. The strategy is based on 13C-13C solution NOESY experiments combined with solid-state proton-driven 13C-13C spin diffusion and 3D coherence transfer experiments. In this way, 75% of amino acids were recognized and 35% sequence-specific assigned. Paramagnetic broadening, induced by iron(III) species in solution 13C-13C NOESY spectra, localized the iron within each subunit and traced the progression to the central cavity. Eight iron ions fill the 20-Å-long iron channel from the ferrous/dioxygen oxidoreductase site to the exit into the cavity, inside the four-helix bundle of each subunit, contrasting with short paths in models. Magnetic susceptibility data support the formation of ferric multimers in the iron channels. Multiple iron channel exits are near enough to facilitate high concentration of iron that can mineralize in the ferritin cavity, illustrating advantages of the multisubunit cage structure.
Keywords:¹  ³  C direct detection, high molecular weight NMR, iron channels, solid-state NMR, paramagnetic NMR
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