Ferritins: furnishing proteins with iron

Ferritins are a superfamily of iron oxidation, storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. The majority of ferritins consist of 24 subunits that individually fold into 4-α-helix bundles and assemble in a highly symmetric manner to form an approximately spherical protein coat around a central cavity into which an iron-containing mineral can be formed. Channels through the coat at inter-subunit contact points facilitate passage of iron ions to and from the central cavity, and intrasubunit catalytic sites, called ferroxidase centers, drive Fe2+ oxidation and O2 reduction. Though the different members of the superfamily share a common structure, there is often little amino acid sequence identity between them. Even where there is a high degree of sequence identity between two ferritins there can be major differences in how the proteins handle iron. In this review we describe some of the important structural features of ferritins and their mineralized iron cores and examine in detail how three selected ferritins oxidise Fe2+ in order to explore the mechanistic variations that exist amongst ferritins. We suggest that the mechanistic differences reflect differing evolutionary pressures on amino acid sequences, and that these differing pressures are a consequence of different primary functions for different ferritins

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation

Characterization of the binding sites of the anticancer ruthenium(III) complexes KP1019 and KP1339 on human serum albumin via competition studies

Indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019) and its Na+ analogue (KP1339) are two of the most prominent non-platinum antitumor metal complexes currently undergoing clinical trials. After intravenous administration, they are known to bind to human serum albumin (HSA) in a noncovalent manner. To elucidate their HSA binding sites, displacement reactions with the established site markers warfarin and dansylglycine as well as bilirubin were monitored by spectrofluorimetry, ultrafiltration-UV-vis spectrophotometry, and/or capillary zone electrophoresis. Conditional stability constants for the binding of KP1019 and KP1339 to sites I and II of HSA were determined, indicating that both Ru(III) compounds bind to both sites with moderately strong affinity (log K (1)' = 5.3-5.8). No preference for either binding site was found, and similar results were obtained for both metal complexes, demonstrating low influence of the counter ion on the binding event

Anion exchange in human serum transferrin N-lobe: a model study with variant His249Ala

The removal of Fe(III) from human serum transferrin by chelators is thought to proceed through intermediate species in which the chelator becomes associated with the metal center of the protein. The visible spectral shifts associated with the formation of such intermediates in the wild-type (WT) protein are too small for reliable kinetic data to be obtained. Therefore, studies were undertaken with the recombinant N-terminal lobe variant H249A, a variant showing more pronounced spectral changes. The kinetics of the synergistic anion-exchange reaction between nitrilotriacetate (NTA) and carbonate in variant H249A was studied by stopped-flow spectrophotometry as a model for this process in the WT protein. Anion exchange occurs by two pathways at pH 7.4 and 25 degrees C: an NTA-independent dissociative pathway to form a carbonate-free intermediate Fe-H249A (Eq. 1) that subsequently reacts with NTA (Eq. 2):and an NTA-dependent associative pathway (the major pathway) in which a quaternary Fe-H249A-(CO(3))(NTA) intermediate is formed (Eq. 3), which then decays to product (Eq. 4):The reverse reaction, where HCO(3)(-) exchanges for NTA, likewise follows these two pathways. The overall apparent equilibrium constant for formation of Fe-H249A-NTA from Fe-H249A-CO(3) is K'=442 at pH 7.4. The NTA complex is favored over the carbonate complex both kinetically and thermodynamically in the pH range 7.4-8.2