Evidence of H- and L-chains have co-operative roles in the iron-uptake mechanism of human ferritin

Abstract

The ability to incorporate iron in vitro was studied in homopolymers of human ferritin L-chain, human ferritin H-chain and its variants and in homopolymer mixtures. The H-chain variants carried amino acid substitutions in the ferroxidase centre and/or in carboxy residues on the cavity surface. Iron incorporation was examined by gel electrophoresis of the reaction products by staining for iron and protein. It was found that inactivation of the ferroxidase centre combined with the substitution of four carboxy groups on the cavity abolished the ability of H-chain ferritin to incorporate iron. Competition experiments with limited amounts of iron showed that, at neutral pH, L-chain ferritin is more efficient in forming iron cores than the H-chain variants altered at the ferroxidase activity or in the cavity. Competition experiments at pH 5.5 demonstrated that L-chain apoferritin is able to incorporate iron only when in the presence of H-chain variants with ferroxidase activity. The results indicate that L-chain apoferritin has a higher capacity than the H-chain apoferritin to induce iron-core nucleation, whereas H-chain ferritin is superior in promoting Fe(II) oxidation. The finding of cooperative roles of the H- and L-chains in ferritin iron uptake provides a clue to understanding the biological function of isoferritins.

Is Hydrogen Peroxide Produced during Iron(II) Oxidation in Mammalian Apoferritins?

“…Titration of apoHoSF revealed very weak catalase activity as evidenced by the slow production of heat, ∆H°) -107 ( 17 kJ/mol, and no end point corresponding to reaction with the protein. 5 Thus, the ITC measurements fully support the findings of the experiments with catalase, namely, that iron has to be present for any reaction with the protein to occur. Also, similar H 2 O 2 reaction stoichiometries are obtained with oximetry and ITC measurements.…”

“…The measured stoichiometry, i.e., end point, is reliable, however. 5 Injections were spaced 20 min apart because of the slow disproportionation reaction observed with the ferritins. Due to the slow return to the baseline following each injection, the measured enthalpies for H2O2 disproportionation may be underestimated by as much as 10%.…”

“…Mammalian ferritins are composed of 24 subunits of two types, H and L, with similar amino acid sequences but distinctly different functions (3). The Hsubunit contains a dinuclear ferroxidase site and appears to be largely involved in promoting Fe(II) oxidation (4) while the more acidic L-subunit is thought to provide an environment for mineralization within the protein cavity (5).…”

Heme Regulation in Traumatic Brain Injury: Relevance to the Adult and Developing Brain

“…Neurons contain abundant H-ferritin, which is important for rapid uptake and reutilization of iron. In contrast, L-ferritin, involved in long-term iron storage, is the dominant form in microglia (Levi et al, 1992). Both L-and H-ferritin are expressed in oligodendrocytes.…”

EPR Studies of Recombinant Horse L-Chain Apoferritin and its Mutant (E 53,56,57,60 Q) with Haemin

“…Following the Drysdale model, the two types of subunits can form 25 different heteropolymers (Drysdale 1977). Hchains catalyse the oxidation of Fe(II) to Fe(III) (ferroxidase activity) and the functional importance of L-chains is thought to reside primarily in their ability to promote nucleation of the ferrihydrite core; it has been demonstrated that these two chains have co-operative roles in iron mobilisation (Bauminger et al 1991;Levi et al 1992;Bauminger et al 1994;Santambrogio et al 1996).…”