Iron Oxidation and Core Formation in Recombinant Heteropolymeric Human Ferritins

Abstract

In animals, the iron storage and detoxification protein, ferritin, is composed of two functionally and genetically distinct subunit types, H (heavy) and L (light), which co-assemble in various ratios with tissue specific distributions to form shell-like protein structures of 24 subunits within which a mineralized iron core is stored. The H-subunit possesses a ferroxidase center (FC) that catalyzes Fe(II) oxidation, whereas the L-subunit does not. To assess the role of the L-subunit in iron oxidation and core formation, two human recombinant heteropolymeric ferritins, designated H-rich and L-rich with ratios of ~20H:4L and ~22L:2H, respectively, were employed and compared to the human homopolymeric H-subunit ferritin (HuHF). These heteropolymeric ferritins have a composition similar to the composition of those found in hearts and brains (i.e., H-rich) and in livers and spleens (i.e., L-rich). As for HuHF, iron oxidation in H-rich ferritin was found to proceed with a 2:1 Fe(II):O2 stoichiometry at an iron level of 2 Fe(II) atoms/H-subunit with the generation of H2O2. The H2O2 reacted with additional Fe(II) in a 2:1 Fe(II):H2O2 ratio, thus avoiding the production of hydroxyl radical. A μ-1,2-peroxo-diFe(III) intermediate was observed at the FC of H-rich ferritin as for HuHF. Importantly, the H-rich protein regenerated full ferroxidase activity more rapidly than HuHF did and additionally formed larger iron cores, indicating dual roles for the L-subunit in facilitating iron turnover at the FC and in mineralization of the core. The L-rich ferritin, while also facilitating iron oxidation at the FC, additionally promoted oxidation at the mineral surface once the iron binding capacity of the FC was exceeded.

Effect of chaotropes on the kinetics of iron release from ferritin by flavin nucleotides

“…Initially, a mechanism based on diffusion of FMNH 2 into the interior of ferritin through either three- or four-fold channels (pores) was proposed. 16 However, multiple recent reports painted a much different picture and casted doubt about the validity of the proposed pore diffusion mechanism in light of the inability of small molecules including cisplatin, 34 anthocyanin, 36 beta-carotene, 37 and curcumin 38 to diffuse through the ferritin pores and be trapped inside. Given the comparable size of flavin mononucleotide to the aforementioned molecules, we explored the ability of FMN to diffuse out of the ferritin pores following its encapsulation inside the protein's cavity.…”

“…Bipyridine, guanidine HCl, and triton X-100 (Sigma-Aldrich), and urea (J.T Baker Chemical Company) were purchased and used as received. Human recombinant heteropolymer apoferritin (∼ 20H- and 4L-subunits) was prepared as described elsewhere 33, 34 and manually loaded with 500 iron atoms in the presence of oxygen in the form of 10 additions of 50 Fe(II)/shell with 10-15 minutes between additions. Unless otherwise stated, all experiments were conducted at 25 °C, in 100 mM MOPS buffer and 50 mM NaCl, pH 7.0.…”

Therapeutic Opportunities for Hepcidin in Acute Care Medicine

“…For instance, the acute-phase proteins include hemopexin, haptoglobin, ferritin, lactoferrin, ceruloplasmin, NGAL, and hepcidin, all of which are involved in iron or iron-complexed macromolecule protective functions. 22,[44][45][46][47][48][49][50] Fig. 4.…”

“…Second, ferritin is a ferroxidase and rapidly converts ferrous (Fe 21 , very toxic) to ferric (Fe 31 , somewhat less toxic) iron. 45,46 Ferric iron is rapidly loaded onto TF in the plasma and ferric iron is also sequestered into ferritin. 46 In general, ferritin is made up of light (L) and heavy (H) subunits.…”

Electromagnetic Regulation of Cell Activity

“…Iron likely enters the shell via channels at the threefold axis and the ferroxidase activity of the H chain catalyzes the oxidation of two Fe(II) ions with the intermediate production of H 2 O 2 . The light chain accelerates iron transport and may generate a more ordered iron oxide core (Bradley et al 2014;Mehlenbacher et al 2017).…”