Iron Biomineral Growth from the Initial Nucleation Seed in L‐Ferritin

Abstract

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...

Iron Binding in the Ferroxidase Site of Human Mitochondrial Ferritin

“…Recent structural evidence, showed that three of these glutamates, namely, Glu60, Glu61 and Glu64 are responsible for anchoring oxo-centered tri-iron(III) clusters on the internal cage surface of HuLf, whereas the fourth glutamate, Glu57, is mainly involved in shuttling the incoming iron ions towards the cluster (Figure 13). [5,6] The position of the Fe5-A site observed in hMTF matches the position of one of the ferric ions (Fe2 in Figure 13) composing the trinuclear cluster in the mineralization site of HuLf.…”

“…The asymmetric unit of hMTF crystals includes a single protein chain, representing the spatial and time average of all protein subunits, as reported previously. [4][5][6]10] Depending on the iron-diffusion time, the structures of hMTF include a variable number of metal ions [Mg(II) and/or Fe(II)/(III) ions] whose chemical nature was discriminated according to the detected anomalous diffraction signal. Iron ions are characterized by a strong signal in the anomalous difference Fourier map determined from data collected with X-ray energy of ~7130 eV (Fe K-edge peak) that disappeared in the map computed from the data collected at energy immediately below this edge.…”

“…[4,10,[17][18][19][20][21] In the structures of iron-treated hMTF, we have observed iron binding analogous to that found in other vertebrate ferritins. [4][5][6]10] A detailed description of iron binding in both types of channels is given as Supporting Information (see Supporting Information-S3.1 and Supporting Information-S3.2; Figures S10 and S11; Tables S2 and S3).…”

“…[1,4] On the other hand, L-type chains are involved in iron biomineralization facilitated through a nucleation site, located on the internal cage surface, in which iron ions assemble as oxo-centered trinuclear iron(III) clusters. [1,5,6] In humans and other mammals, a further type of ferritin has been identified, namely the mitochondrial ferritin (MTF). Human MTF (hMTF), encoded by an intronless gene, is expressed as a precursor protein having a N-terminal signal peptide sequence which mediates mitochondria localization.…”

On the Role of Poly-Glutamic Acid in the Early Stages of Iron(III) (Oxy)(hydr)oxide Formation

“…Nucleation of inorganic minerals in the presence of (bio)macromolecules increasingly attracts research attention owing to the possibility of progression toward controlling the synthesis of advanced materials and the understanding of biomineralization processes. Iron (oxy)(hydr)oxides are important (bio)-minerals [1], and their nucleation and crystal growth are closely related to the presence of bio-macromolecules in natural systems [2][3][4]. There are multiple roles that additives can play, while it has already been well established that nucleation in the presence of some additives, especially in the early stages of mineralization, cannot be satisfactorily explained from the viewpoint of classical nucleation theory [5].…”