Mineralization in Ferritin: An Efficient Means of Iron Storage N. Dennis Chasteen* and Pauline M. Harrison† *Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824; and †Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2TN, United Kingdom Received January 21, 1999 Ferritins are a class of iron storage and mineraliza- tion proteins found throughout the animal, plant, and microbial kingdoms. Iron is stored within the protein shell of ferritin as a hydrous ferric oxide nanoparticle with a structure similar to that of the mineral ‘‘ferrihydrite.’’ The eight hydrophilic chan- nels that traverse the protein shell are thought to be the primary avenues by which iron gains entry to the interior of eukaryotic ferritins. Twenty-four subunits constitute the protein shell and, in mamma- lian ferritins, are of two types, H and L, which have complementary functions in iron uptake. The H chain contains a dinuclear ferroxidase site that is located within the four-helix bundle of the subunit; it catalyzes the oxidation of ferrous iron by O 2 , producing H 2 O 2 . The L subunit lacks this site but contains additional glutamate residues on the inte- rior surface of the protein shell which produce a microenvironment that facilitates mineralization and the turnover of iron(III) at the H subunit ferroxi- dase site. Recent spectroscopic studies have shown that a di-Fe(III) peroxo intermediate is produced at the ferroxidase site followed by formation of a -oxo- bridged dimer, which then fragments and migrates to the nucleation sites to form incipient mineral core species. Once sufficient core has developed, iron oxidation and mineralization occur primarily on the surface of the growing crystallite, thus mini- mizing the production of potentially harmful H 2 O 2 .  1999 Academic Press Key Words: ferrihydrite; ferritin; ferritin struc- tures; iron core; iron mineralization; iron storage I. INTRODUCTION In bones and shells, biomineralization is used to provide strength or protection to an organic scaffold and the mineral is extracellular. In ferritin the situation is different: the mineral is sequestered within a single molecule, which has a protein shell of defined size and form. The ferritin protein shell has several functions: it acquires iron(II), catalyses its oxidation, and induces mineralization within its cavity. Thus nonspecific iron(III) hydrolysis is avoided. Ferritin also influences the mineral form in which iron(III) is stored within it, and its soluble protein coat prevents the uncontrolled growth and coalescence of the small mineral particles into larger insoluble aggregates. The protein shell also limits the accessibility of cell constituents to the iron mineral. Iron-free ferritin molecules (apoferritin) are hol- low spheres with an outer diameter of 12 nm, and an inner diameter of 8 nm and have a molecular weight about half a million (Lawson et al., 1991; Harrison and Arosio, 1996). They are composed of 24 protein chains or subunits arranged in 432 symmetry (Fig. 1a). The three-dimensional structure is very well conserved throughout the animal and indeed the plant and microbial kingdoms. The protein shell can house up to 4500 iron atoms within its 8-nm- diameter cavity (Fig. 1b). Such a high Fe:protein ratio (200 times that in hemoglobin) is made possible by sequestering the iron as a compact mineral (Fig. 2). The fact that mineralization occurs within pre- formed intact shells, which limit the size of the hydrous ferric oxide particles, may also influence the availability of the iron. Very little is known about the chemical nature of the iron reaching ferritin in vivo. There is very little free iron(II) within cells (about 10 -8 M (Williams, 1982) and an abundance of potential chelators such as citrate. Nevertheless, controlling the iron mineral- ization of ferritin shells is accomplished through the matching of protein concentration to cellular iron levels. This is achieved in both eukaryotes and prokaryotes by the response of the protein’s biosyn- thetic apparatus to cellular iron (reviewed in Hentze and Ku ¨ hn, 1996). Here we discuss our current knowledge of the structure of the iron mineral and the ferritin protein shell and our understanding of the steps leading to iron mineralization. The emphasis will be on animal ferritins, most of the information being derived from human, horse, and frog proteins. Bacterial ferritins Journal of Structural Biology 126, 182–194 (1999) Article ID jsbi.1999.4118, available online at http://www.idealibrary.com on 182 1047-8477/99 $30.00 Copyright  1999 by Academic Press All rights of reproduction in any form reserved.