Chemistry at the protein-mineral interface: the nucleation site of iron mineral in human L ferritin revealed by anomalous x-ray diffraction

Iron ions have crucial functions in every living organism being essential for cellular respiration, DNA synthesis, detoxification of exogenous compounds, just to provide a few examples. However, the redox properties of iron ions can also cause the occurrence of deleterious free-radicals. For these reasons, when unnecessary, iron must be kept in appropriate forms unable to cause damage. Nature evolved a special protein cage, called ferritin, consisting of 24 subunits arranged to form a hollow sphere with an internal diameter of about 80 Å where mineralized iron is stored, generally under the form of insoluble ferric oxides. In mammals, two types of subunits build-up the 24-mer ferritins: the ‘heavy’ (H) and the ‘light’ (L). These subunits differ not only in molecular weight (21.2 kDa for H and 20.0 kDa for L) but, mainly, in function. The H subunit is able to catalyze the rapid oxidation of Fe2+ to Fe3+ followed by transfer in the storage cavity. On the contrary, the L-chain does not possess catalytic activity, but it is still able to mineralize ferric ions upon spontaneous oxidation by dioxygen of captured Fe2+. Despite the intensive research on ferritin chemistry, the mechanisms of iron oxidation and storage to form mineral nanoparticles inside the ferritin cavity are still to be fully established.
We performed two-wavelength anomalous diffraction measurements around the iron K-edge on L-ferritin crystals treated with ferrous salt and frozen at different times after exposure to Fe2+ ions. The experiment has been performed at the Elettra beamline XRD-1 and complemented by high energy data collection at the Diamond Light Source (DLS; UK). The crystal structure revealed the occurrence of a beautiful oxo-centered trinuclear Fe3+ cluster (Figure 1) anchored to the protein through glutamic acid side chains. The three Fe3+ ions are held by an oxide anion in the center and bridge two adjacent Glu side chains. The residual electron density bridging each couple of Fe3+ ions is assigned to three peroxide molecule originated by Fe2+ oxidation by dioxygen. An incoming fourth Fe3+ ion is approaching the cluster shuttled by a fourth nearby Glu residue. Remarkably, the iron cluster observed in L-ferritin has the same structure of half the hexanuclear Fe3+ cluster reported twenty years ago (J Am Chem Soc 1997, 119: 1037) as a model for the ferritin mineral core (Figure 2). The presence of peroxide anions bound to the cluster has been confirmed by UV-vis spectroscopy and the functional role of the three adjacent glutamate residues (Glu60, Glu61, Glu64) as the nucleating center for mineralization has been established by kinetic studies on the triple variant E60AE61AE64A. The same trinuclear cluster has been found by an analogous experiment performed on horse spleen ferritin crystals.
This work has established for the first time the mineral nucleation site in L-type ferritins and revealed the iron mineral nucleus and the mechanism of mineral formation that differs from that occurring in H-type ferritins, where the peculiar arrangement of the three Glu residues is absent. Indeed, iron clusters have never been detected in H-type ferritins by crystallographic experiments despite the numerous attempts made by us.
This study demonstrates, once more, the usefulness of synchrotron time-lapse cryo-crystallography to trap and reveal the structure of reaction intermediates in ferritins, allowing the identification of functional iron-binding sites and of the dynamical processes that span from iron entering the cages to mineral formation.
In a more general view, this kind of studies can shed light on the many still unknown process occurring at the protein-mineral interface.

Figure 1. The iron cluster present in human L-ferritin revealed by time-lapse anomalous crystallography. The anomalous difference electron density map calculated from data collected at Elettra (beamline XRD-1) at energy above and below the Fe K-edge is superimposed to the Fe3+ ions (cyan spheres).


Figure 2. Crystal structure of the synthetic model of ferritin mineral. The tris(µ4-peroxo) bis(µ3-oxo) nonakis(µ2-ac) hexa-iron(II) complex obtained by Shweky et al. (J. Am. Chem. Soc. 1997, 119, 1037). The figure shows only half of the exanuclear iron cluster to highlight the equivalence of the model to the L-ferritin cluster. 


This research was conducted by the following research team:

Cecilia Pozzi1, Flavio Di Pisa1, Stefano Mangani1, Silvia Ciambellotti2, Caterina Bernacchioni2, Paola Turano2


1 Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy
2 CERM and Department of Chemistry, University of Florence, Sesto Fiorentino, Firenze, Italy

Contact person:

Maurizio Polentarutti, email:



Cecilia Pozzi, Silvia Ciambellotti, Caterina Bernacchioni, Flavio Di Pisa, Stefano Mangani, PaolaTurano “Chemistry at the protein-mineral interface in L-ferritin assists the assembly of a functional (μ3-oxo)Tris[(μ2-peroxo)] triiron(III) cluster” Proc. Natl. Acad. Sci. USA. (2017) pii: 201614302. doi: 10.1073/pnas.1614302114
Last Updated on Monday, 27 March 2017 15:57