Ferritin, iron homeostasis, and oxidative damage1, 2

Abstract

Ferritin is one of the major proteins of iron metabolism. It is almost ubiquitous and tightly regulated by the metal. Biochemical and structural properties of the ferritins are largely conserved from bacteria to man, although the role in the regulation of iron trafficking varies in the different organisms. Recent studies have clarified some of the major aspects of the reaction between iron and ferritin, which results in the formation of the iron core and production of hydrogen peroxide. The characterization of cellular models in which ferritin expression is modulated has shown that the ferroxidase catalytic site on the H-chain has a central role in regulating iron availability. In turn, this has secondary effects on a number of cellular activities, which include proliferation and resistance to oxidative damage. Moreover, the response to apoptotic stimuli is affected by H-ferritin expression. Altered ferritin L-chain expression has been found in at least two types of genetic disorders, although its role in the determination of the pathology has not been fully clarified. The recent discovery of a new ferritin specific for the mitochondria, which is functionally similar to the H-ferritin, opens new perspectives in the study of the relationships between iron, oxidative damage and free radicals.

Introduction

Ferritin is a nanobox protein designed to contain and maintain in solution up to a few thousand iron atoms, which otherwise would aggregate in toxic precipitates. Ferritins originated early in phylogenesis and are present in archeobacteria, eubacteria, plants, invertebrates, and mammals. Ferritin genes are present, often in multiple copies, in most organisms, with the notable exception of yeast, which uses alternative mechanisms to store excess iron. Although ferritins from different origin may have largely different sequences, with identities as low as 15%, their three-dimensional structures are remarkably highly conserved. The peptide chains fold into four helix bundles with a long loop between helices B and C and a fifth short helix at the C-terminus (Fig. 1A). Twenty-four subunits assemble in large, highly stable complexes with 2,3,4 point symmetry (Fig. 1B). Crystallographic structures have been resolved for ferritins from Escherichia coli, both the heme bacterioferritin BFR and the nonheme FTN, two forms of bullfrog ferritins, and mammalian ferritins (human, horse, and mouse) (reviewed in [1]). They are all very similar, as illustrated by the fact that the α carbons of the E. coli and human ferritins superimpose within 0.2 nm. More recently, a new form of bacterial ferritin has been identified in Listeria innocua, which is composed of subunits with similar fold but that assemble in a 12-mer complex [2]. It is structurally analogous to the DNA protection during starvation protein (DPS) of E. coli, a DNA binding molecule that is expressed under stress conditions. The typical 24-mer ferritins have an almost spherical shape that delimits a large cavity about 8 nm across, which can accommodate up to 4000 Fe atoms in a ferric hydroxide core, the presence of which does not affect protein surface and possible interactions with other molecules. The proteins are also characterized by catalytic sites for iron oxidation and hydrophilic pores for exchange with the solvent.

All the ferritins have the property to interact readily with Fe(II) ions in solution under aerobic conditions and to induce iron oxidation and aggregation inside the cavity. This process has been carefully studied and is critically reviewed in ref [1]. Essentially, the reaction with iron initiates with the binding of Fe(II) to a specific site located inside the subunit helical fold, named ferroxidase center. Iron interacts with oxygen, is oxidized to Fe(III) and then migrates to the cavity where it nucleates and aggregates to form the iron core. In in vitro reactions, the oxidation step is fast, being completed in seconds, while the hydrolysis-mineralization step, which determines the turnover of the ferroxidase site, is slower and completed in minutes. The seven residues that contribute to the formation of the ferroxidase site are conserved in all ferritins, the exception being the L-subunits of vertebrate ferritins (see later). However, some differences have been observed in the mechanisms of iron oxidation in different ferritins. For example, the FTN ferritin from E. coli oxidizes iron more slowly in a reaction that produces water [3], while human H-ferritin oxidizes iron more readily in a reaction with produces hydrogen peroxide [4]. The formation of this product in the vicinity of iron is potentially harmful because it would initiate Fenton’s reaction, and this may be relevant for processes of protein damage and the formation of hemosiderin, a putative insoluble degradation product of ferritin.

The ferritins from bacteria and plants are composed of 24 subunit of the same type, thus they have 24 catalytic sites that concur to the formation of one or two iron cores per molecule. The vertebrates have a second, L-type subunit that coassemble with the catalytically active H-chain to form heteropolymers. In this subunit the residues of the ferroxidase center are substituted with the formation of an intra-chain salt bridge that further stabilizes the protein [5]. The peculiarity of this subunit is to offer acidic residues on the surface cavity that facilitate iron nucleation and increase the turnover of the ferroxidase site. Thus, the hybrid molecules composed by H- and L-chains are more efficient in taking up iron than the H homopolymers [6]. It should be noticed that the presence of two subunit types with specific functionality that can associate in any proportion offers the possibility to regulate independently the number of ferritins molecules, i.e., the iron storage capacity, and the number of catalytic sites, i.e., the iron oxidizing capacity. This is particularly evident in tissues with iron storage function, such as liver and spleen, or in tissues with high iron load, where the production of a large number of L-chains allows the cell to expand its iron storage capacity without an excess of catalytic sites, which would limit iron availability.

Ferritins are mainly cytosolic proteins and keep the stored iron separated from the nucleus and other organelles. However, a minor proportion of ferritin in vertebrates is present also in serum and secretory fluids. Serum ferritin is a clinically important index of body iron stores, but its functional role remains largely obscure. However, secretory ferritins are found in some insects and mollusks, and they may have an important role in releasing iron from the cell [1]. Probably more intriguing is the observation that plant ferritins are specifically localized inside the plastids [7]. These organelles are rich in iron enzymes for photosynthesis and are also exposed to strong oxidative stress. This suggests that ferritin may have a protective role, however, the overexpression of ferritin in transgenic plants was found to induce a relative iron starvation and increase the sensitivity to oxidative damage [8].