Iron binding to human heavy-chain ferritin

Abstract

Maxi-ferritins are ubiquitous iron-storage proteins with a common cage architecture made up of 24 identical subunits of five α-helices that drive iron biomineralization through catalytic iron(II) oxidation occurring at oxidoreductase sites (OS). Structures of iron-bound human H ferritin were solved at high resolution by freezing ferritin crystals at different time intervals after exposure to a ferrous salt. Multiple binding sites were identified that define the iron path from the entry ion channels to the oxidoreductase sites. Similar data are available for another vertebrate ferritin: the M protein from Rana catesbeiana. A comparative analysis of the iron sites in the two proteins identifies new reaction intermediates and underlines clear differences in the pattern of ligands that define the additional iron sites that precede the oxidoreductase binding sites along this path. Stopped-flow kinetics assays revealed that human H ferritin has different levels of activity compared with its R. catesbeiana counterpart. The role of the different pattern of transient iron-binding sites in the OS is discussed with respect to the observed differences in activity across the species.

Chemistry at the protein–mineral interface in L-ferritin assists the assembly of a functional (μ 3 -oxo)Tris[(μ 2 -peroxo)] triiron(III) cluster

“…1B). Consistently, the biomineralization reaction in H-ferritin cages does not require a nucleation site (24,25), and His57 and Glu61 are involved in controlling the access of ferrous ions to the ferroxidase site (6); the reaction at the ferroxidase site is much faster than in L-subunits, with time scales for the iron oxidation in homopolymeric H-cages of <1 s. Even with a low number of H-subunits (1 to 2 per cage), the reaction rate is dominated by the catalytic oxidation of iron occurring at the ferroxidase sites. The formation of ferric clusters of high nuclearity in ferroxidase-active (H or H′) subunits has been inferred from Mössbauer data (26)(27)(28) and NMR data (29,30), but these clusters have never been detected by time-lapse anomalous crystallography approaches.…”

“…In H-type subunits, like human H-ferritin (HuHf), this ideal arrangement of carboxylate side chains is disrupted by replacement of Glu60 with a His, with the concomitant increase in the conformational freedom of the Glu61 side chain (6). Also, Glu57 is replaced by His in HuHf (Fig.…”

“…A series of L-ferritin structures were determined by soaking HuLF and HoLf ferritin crystals with a freshly prepared [(NH 4 ) 2 Fe(SO 4 ) 2 ]•6H 2 O solution for exposure times of 15 to 60 min, followed by flash freezing in liquid nitrogen. The procedure, detailed in SI Materials and Methods, follows with some modifications an established protocol that already allowed us to characterize iron oxidation in the ferroxidase site of human H-and Rana catesbeiana H′-type ferritins (4,6). Data collection, structure solution, and refinement procedures are given in SI Materials and Methods and in Tables S2 and S3.…”

“…In the human H-subunit, Glu62 acts as a bridging ligand between the two metals; the metal at Fe1 is also bound to His65 and monodentate Glu27 whereas the metal at Fe2 binds bidentate Glu107 (4). Accessory transient metal sites (Fe3, Fe4, and, in some cases, Fe5) have been identified by X-ray crystallography in the proximity of the ferroxidase site, and they have been demonstrated to play a key role in the reaction turnover (4,6). L-subunits lack the ferroxidase site whereas a number of other residues are conserved with respect to the H-subunit (homology 53% between human L-and H-chains (Fig.…”

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels

“…This experimental evidence has suggested that the presence of carboxylates at the inner edge of wild-type C3 channels, as well as engineered at the inner edge of C4 channels, determines a favorable electric field for driving Fe 2ϩ into the cage. Accordingly, high-resolution X-ray crystal structures (12,13) have shown two ferrous hexa-aqua ions within vertebrate C3 channels, but only one iron ion within the C4 channels coordinated by four His-169 N⑀2, a water molecule, and a chloride anion (see Fig. 1C).…”