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.

X-ray structures of homopolymeric L-ferritin obtained by freezing protein crystals at increasing exposure times to a ferrous solution showed the progressive formation of a triiron cluster on the inner cage surface of each subunit. After 60 min exposure, a fully assembled (μ 3 -oxo)Tris[(μ 2 -peroxo)(μ 2 -glutamato-κO:κO′)](glutamato-κO)(diaquo)triiron(III) anionic cluster appears in human L-ferritin. Glu60, Glu61, and Glu64 provide the anchoring of the cluster to the protein cage. Glu57 shuttles incoming iron ions toward the cluster. We observed a similar metallocluster in horse spleen L-ferritin, indicating that it represents a common feature of mammalian L-ferritins. The structures suggest a mechanism for iron mineral formation at the protein interface. The functional significance of the observed patch of carboxylate side chains and resulting metallocluster for biomineralization emerges from the lower iron oxidation rate measured in the E60AE61AE64A variant of human L-ferritin, leading to the proposal that the observed metallocluster corresponds to the suggested, but yet unobserved, nucleation site of L-ferritin.L-ferritin | metallocluster | nucleation site | biomineralization | X-ray

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.

The aim of this work is to identify the cisplatin binding sites on human H-chain ferritin. High-resolution X-ray crystallography reveals that cisplatin binds four distinct protein sites, that is, the side chains of His136 and Lys68, the side chain of His105, the side chain of Cys90 and the side chain of Cys102. These Pt binding sites are compared with those observed for the adduct that cisplatin forms upon encapsulation within horse spleen L-chain ferritin (87% identity with human L-chain ferritin).

X-ray structures of homopolymerich uman L-ferritin andh orse spleen ferritin weres olved by freezing protein crystals at different time intervals after exposure to a ferric salt and revealed the growth of an octa-nuclear iron cluster on the inner surface of the protein cage with ak ey role played by some glutamate residues.A na tomic resolution view of how the clusterf ormation developss tarting from a( m 3 -oxo)tris[(m 2 -glutamato-kO:kO')](glutamato-kO)-(diaquo)triiron(III) seed is provided. The results support the idea that iron biomineralizationi nf erritin is ap rocess initiating at the level of the protein surface, capable of contributing coordinationb onds and electrostatic guidance.In animals, cytosolic ferritin is ah eteropolymer consisting of 24 subunits of H-and L-chains that self-assemble into ah ollow structure that hosts iron deposits. L-subunits lack the ferroxidase site for the catalytic oxidation of Fe 2 + ,w hich is the characteristic feature of H-subunits. [1,2] The H/L ratio in the heteropolymeri sd etermined by the different expression levels of these two components and is tissue and cell specific. [3,4] In humans,c ages rich in Hs ubunits are found in tissues requiring fast iron metabolism (e.g.,m uscles and heart), whereas cages rich in Ls ubunits are found in tissues involved in long-term iron storage (such as liver and spleen). In brain, neurons express mostly H-ferritin, microglia express mostly L-ferritin, oligodendrocytes express similar amounts of both Ha nd Ls ubunits. [5] The H/L ratio determines the rate of iron biomineralization, which occurs as the result of the formation of nanosized particles of iron oxidesw ithin the inner nanocage cavity. [6] From TEM, SAXS, XANES, EELS and SQUID data, [7][8][9][10] ap olypha-sic structure (ferrihydrite,m agnetite, hematite) of the bulk biomineral has been proposed,w ith af errihydrite enriched core and ap redominantly magnetite-like surface. [7] The amount of L-type subunits influences the morphology,a se videnced by STEM micrographs, giving rise to hollow structures with shapes defined by the number of nucleation sites in the protein shell. [8] Nevertheless, the atomic-levelm echanism of the mineral formationr emains elusive. Recently,b yu sing timelapse crystallographic techniques, we have observed biomineral seeds consistingo f( m 3 -oxo)tris[(m 2 -peroxo)] triiron(III) clusters at the protein-inner cavity interface, which form upon spontaneous oxidation of ferrous ions internalized by recombinant human homopolymeric L-ferritin as well as by natural horse spleen ferritin, which contains about 1-2Hsubunits/cage (Figures S1 Aa nd S2 A, Supporting Information). [11] In this work, we have modified our experimental setting to extendt he observation beyond the formation of these initial nucleation clusters. In the previouse xperiments, we could observe Fe 3 + clusterf ormation via free diffusion of Fe 2 + through L-ferritin crystalsf ollowed by spontaneous oxidation, and flash freezing. [11] Undert hose experimental conditions, in the homopolym...

The reaction of the antimetastatic ruthenium(iii) drug NAMI A with human H-chain ferritin (HuHf) was investigated through a variety of biophysical methods. We observed that the addition of HuHf to NAMI A solutions significantly increases the rate of spontaneous NAMI A hydrolysis suggesting the occurrence of a direct metallodrug-protein interaction. The resulting hydrolyzed Ru species binds the protein mostly forming a relatively tight 1 : 1 ruthenium/ferritin (subunit) adduct that was then separated and characterized. Notably, this adduct shows a characteristic CD spectrum in the visible region, which is diagnostic of the existence of at least one protein bound ruthenium center. The crystal structure of this NAMI A/HuHf adduct was subsequently solved at 1.58 Å resolution; clear evidence is given for the selective binding of a single Ru ion to His105 of each subunit with concomitant release of all other original Ru ligands in agreement with previous observations. We also noted that NAMI A produces a partial inhibition of HuHf ferroxidase activity. The implications of the above results are discussed.

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We investigated the kinetics of the release of iron(II) ions from the internal cavity of human H-ferritin as a function of pH. Extensive molecular dynamics simulations of the entire 24-mer ferritin provided atomic-level information on the release mechanism. Double protonation of His residues at pH 4 facilitates the removal of the iron ligands within the C3 channel through the formation of salt bridges, resulting in a significantly lower release energy barrier than pH 9.