The iron redox and hydrolysis chemistry of the ferritins

Background

Ferritins are ubiquitous and well-characterized iron storage and detoxification proteins. In bacteria and plants, ferritins are homopolymers composed of H-type subunits, while in vertebrates, they typically consist of 24 similar subunits of two types, H and L. The H-subunit is responsible for the rapid oxidation of Fe(II) to Fe(III) at a dinuclear center, whereas the L-subunit appears to help iron clearance from the ferroxidase center of the H-subunit and support iron nucleation and mineralization.

Scope of review

Despite their overall similar structures, ferritins from different origins markedly differ in their iron binding, oxidation, detoxification, and mineralization properties. This chapter provides a brief overview of the structure and function of ferritin, reviews our current knowledge of the process of iron uptake and mineral core formation, and highlights the similarities and differences of the iron oxidation and hydrolysis chemistry in a number of ferritins including those from archaea, bacteria, amphibians, and animals.

General Significance

Prokaryotic ferritins and ferritin-like proteins (Dps) appear to preferentially use H₂O₂ over O₂ as the iron oxidant during ferritin core formation. While the product of iron oxidation at the ferroxidase centers of these and other ferritins is labile and is retained inside the protein cavity, the iron complex in the di-iron cofactor proteins is stable and remains at the catalytic site. Differences in the identity and affinity of the ferroxidase center ligands to iron have been suggested to influence the distinct reaction pathways in ferritins and the di-iron cofactor enzymes.

Major conclusions

The ferritin 3-fold channels are shown to be flexible structures that allow the entry and exit of different ions and molecules through the protein shell. The H- and L-subunits are shown to have complementary roles in iron oxidation and mineralization, and hydrogen peroxide appears to be a by-product of oxygen reduction at the FC of most ferritins. The di-iron(III) complex at the FC of some ferritins acts as a stable cofactor during iron oxidation rather than a catalytic center where Fe(II) is oxidized at the FC followed by its translocation to the protein cavity.     

Could a dysfunction of ferritin be a determinant factor in the aetiology of some neurodegenerative diseases?

Background

The concentration of iron in the brain increases with aging. Furthermore, it has also been observed that patients suffering from neurological diseases (e.g. Parkinson, Alzheimer…) accumulate iron in the brain regions affected by the disease. Nevertheless, it is still not clear whether this accumulation is the initial cause or a secondary consequence of the disease. Free iron excess may be an oxidative stress source causing cell damage if it is not correctly stored in ferritin cores as a ferric iron oxide redox-inert form.

Scope

Both, the composition of ferritin cores and their location at subcellular level have been studied using analytical transmission electron microscopy in brain tissues from progressive supranuclear palsy (PSP) and Alzheimer disease (AD) patients.

Major conclusions

Ferritin has been mainly found in oligodendrocytes and in dystrophic myelinated axons from the neuropili in AD. In relation to the biomineralization of iron inside the ferritin shell, several different crystalline structures have been observed in the study of physiological and pathological ferritin. Two cubic mixed ferric–ferrous iron oxides are the major components of pathological ferritins whereas ferrihydrite, a hexagonal ferric iron oxide, is the major component of physiological ferritin. We hypothesize a dysfunction of ferritin in its ferroxidase activity.

General significance

The different mineralization of iron inside ferritin may be related to oxidative stress in olygodendrocites, which could affect myelination processes with the consequent perturbation of information transference.     

Phytoferritin and its implications for human health and nutrition

Background

Plant and animal ferritins stem from a common ancestor, but plant ferritins exhibit various features that are different from those of animal ferritins. Phytoferritin is observed in plastids (e.g., chloroplasts in leaves, amyloplasts in tubers and seeds), whereas animal ferritin is largely found in the cytoplasm. The main difference in structure between plant and animal ferritins is the two specific domains (TP and EP) at the N-terminal sequence of phytoferritin, which endow phytoferritin with specific iron chemistry. As a member of the nonheme iron group of dietary iron sources, phytoferritin consists of 24 subunits that assemble into a spherical shell storing up to ∼ 2000 Fe^3 + in the form of an iron oxyhydroxide-phosphate mineral. This feature is distinct from small molecule nonheme iron existing in cereals, which has poor bioavailability.

Scope of review

This review focuses on the relationship between structure and function of phytoferritin and the recent progress in the use of phytoferritin as iron supplement.

Major conclusions

Phytoferritin, especially from legume seeds, represents a novel alternative dietary iron source.

General significance

An understanding of the chemistry and biology of phytoferritin, its interaction with iron, and its stability against gastric digestion is beneficial to design diets that will be used for treatment of global iron deficiency.     

Stability and iron oxidation properties of a novel homopolymeric plant ferritin from adzuki bean seeds: A comparative analysis with recombinant soybean seed H-1 chain ferritin

Background

All reported plant ferritins are heteropolymers comprising two different H-type subunits. Whether or not homopolymeric plant ferritin occurs in nature is an open question.

Methods

A homopolymeric phytoferritin from adzuki bean seeds (ASF) was obtained by various protein purification techniques for the first time, which shares the highest identity (89.6%) with soybean seed H-1 ferritin (rH-1). Therefore, we compared iron oxidation activity and protein stability of ASF with those of rH-1 by stopped-flow combined with light scattering or UV/Vis spectrophotography, SDS- and native- PAGE analyses. Additionally, a new rH-1 variant (S68E) was prepared by site-directed mutagenesis approach to elucidate their difference in protein stability.

Results

At high iron loading of protein, the extension peptide (EP) of plant ferritin was involved in iron oxidation, and the EP of ASF exhibited a much stronger iron oxidative activity than that of rH-1. Besides, ASF is more stable than rH-1 during storage, which is ascribed to one amino acid residue, Ser68.

Conclusions

ASF exhibits a different mechanism in iron oxidation from rH-1 at high iron loading of protein, and a higher stability than rH-1. These differences are mainly stemmed from their different EP sequences.

General significance

This work demonstrates that plant cells have evolved the EP of phytoferritin to control iron chemistry and protein stability by exerting a fine tuning of its amino acid sequence.     

Iron core mineralisation in prokaryotic ferritins

Background

To satisfy their requirement for iron while at the same time countering the toxicity of this highly reactive metal ion, prokaryotes have evolved proteins belonging to two distinct sub-families of the ferritin family: the bacterioferritins (BFRs) and the bacterial ferritins (Ftns). Recently, Ftn homologues have also been identified and characterised in archaeon species. All of these prokaryotic ferritins function by solubilising and storing large amounts of iron in the form of a safe but bio-available mineral.

Scope of review

The mechanism(s) by which the iron mineral is formed by these proteins is the subject of much current interest. Here we review the available information on these proteins, with particular emphasis on significant advances resulting from recent structural, spectroscopic and kinetic studies.

Major conclusions

Current understanding indicates that at least two distinct mechanisms are in operation in prokaryotic ferritins. In one, the ferroxidase centre acts as a true catalytic centre in driving Fe²⁺ oxidation in the cavity; in the other, the centre acts as a gated iron pore by oxidising Fe²⁺ and transferring the resulting Fe³⁺ into the central cavity.

General significance

The prokaryotic ferritins exhibit a wide variation in mechanisms of iron core mineralisation. The basis of these differences lies, at least in part, in structural differences at and around the catalytic centre. However, it appears that more subtle differences must also be important in controlling the iron chemistry of these remarkable proteins.     

Serum ferritin: Past,present and future

Background

Serum ferritin was discovered in the 1930s, and was developed as a clinical test in the 1970s. Many diseases are associated with iron overload or iron deficiency. Serum ferritin is widely used in diagnosing and monitoring these diseases.

Scope of review

In this chapter, we discuss the role of serum ferritin in physiological and pathological processes and its use as a clinical tool.

Major conclusions

Although many aspects of the fundamental biology of serum ferritin remain surprisingly unclear, a growing number of roles have been attributed to extracellular ferritin, including newly described roles in iron delivery, angiogenesis, inflammation, immunity, signaling and cancer.

General significance

Serum ferritin remains a clinically useful tool. Further studies on the biology of this protein may provide new biological insights.     

Iron release from ferritin by flavin nucleotides

Background

Extensive in-vitro studies have focused on elucidating the mechanism of iron uptake and mineral core formation in ferritin. However, despite a plethora of studies attempting to characterize iron release under different experimental conditions, the in-vivo mobilization of iron from ferritin remains poorly understood.Several iron-reductive mobilization pathways have been proposed including, among others, flavin mononucleotides, ascorbate, glutathione, dithionite, and polyphenols. Here, we investigate the kinetics of iron release from ferritin by reduced flavin nucleotide, FMNH2, and discuss the physiological significance of this process in-vivo.

Methods

Iron release from horse spleen ferritin and recombinant human heteropolymer ferritin was followed by the change in optical density of the Fe(II)–bipyridine complex using a Cary 50 Bio UV–Vis spectrophotometer. Oxygen consumption curves were followed on a MI 730 Clark oxygen microelectrode.

Results

The reductive mobilization of iron from ferritin by the nonenzymatic FMN/NAD(P)H system is extremely slow in the presence of oxygen and might involve superoxide radicals, but not FMNH2. Under anaerobic conditions, a very rapid phase of iron mobilization by FMNH2 was observed.

Conclusions

Under normoxic conditions, FMNH2 alone might not be a physiologically significant contributor to iron release from ferritin.

General significance

There is no consensus on which iron release pathway is predominantly responsible for iron mobilization from ferritin under cellular conditions. While reduced flavin mononucleotide (FMNH2) is one likely candidate for in-vivo ferritin iron removal, its significance is confounded by the rapid oxidation of the latter by molecular oxygen.     

Oxido-reduction is not the only mechanism allowing ions to traverse the ferritin protein shell

Background

Most models for ferritin iron release are based on reduction and chelation of iron. However, newer models showing direct Fe(III) chelation from ferritin have been proposed. Fe(III) chelation reactions are facilitated by gated pores that regulate the opening and closing of the channels.

Scope of review

Results suggest that iron core reduction releases hydroxide and phosphate ions that exit the ferritin interior to compensate for the negative charge of the incoming electrons. Additionally, chloride ions are pumped into ferritin during the reduction process as part of a charge balance reaction. The mechanism of anion import or export is not known but is a natural process because phosphate is a native component of the iron mineral core and non-native anions have been incorporated into ferritin in vitro. Anion transfer across the ferritin protein shell conflicts with spin probe studies showing that anions are not easily incorporated into ferritin. To accommodate both of these observations, ferritin must possess a mechanism that selects specific anions for transport into or out of ferritin. Recently, a gated pore mechanism to open the 3-fold channels was proposed and might explain how anions and chelators can penetrate the protein shell for binding or for direct chelation of iron.

Conclusions and general significance

These proposed mechanisms are used to evaluate three in vivo iron release models based on (1) equilibrium between ferritin iron and cytosolic iron, (2) iron release by degradation of ferritin in the lysosome, and (3) metallo-chaperone mediated iron release from ferritin.     

Iron homeostasis and eye disease

Background

Iron is necessary for life, but excess iron can be toxic to tissues. Iron is thought to damage tissues primarily by generating oxygen free radicals through the Fenton reaction.

Methods

We present an overview of the evidence supporting iron's potential contribution to a broad range of eye disease using an anatomical approach.

Results

Iron can be visualized in the cornea as iron lines in the normal aging cornea as well as in diseases like keratoconus and pterygium. In the lens, we present the evidence for the role of oxidative damage in cataractogenesis. Also, we review the evidence that iron may play a role in the pathogenesis of the retinal disease age-related macular degeneration. Although currently there is no direct link between excess iron and development of optic neuropathies, ferrous iron's ability to form highly reactive oxygen species may play a role in optic nerve pathology. Lastly, we discuss recent advances in prevention and therapeutics for eye disease with antioxidants and iron chelators.

General significance

Iron homeostasis is important for ocular health.     

The role of frataxin in fission yeast iron metabolism: Implications for Friedreich's ataxia

Background

The neurodegenerative disease Friedreich's ataxia is the result of frataxin deficiency. Frataxin is a mitochondrial protein involved in iron–sulfur cluster (Fe–S) cofactor biogenesis, but its functional role in this pathway is debated. This is due to the interconnectivity of iron metabolic and oxidative stress response pathways that make distinguishing primary effects of frataxin deficiency challenging. Since Fe–S cluster assembly is conserved, frataxin overexpression phenotypes in a simple eukaryotic organism will provide additional insight into frataxin function.

Methods

The Schizosaccharomyces pombe frataxin homologue (fxn1) was overexpressed from a plasmid under a thiamine repressible promoter. The S. pombe transformants were characterized at several expression strengths for cellular growth, mitochondrial organization, iron levels, oxidative stress, and activities of Fe–S cluster containing enzymes.

Results

Observed phenotypes were dependent on the amount of Fxn1 overexpression. High Fxn1 overexpression severely inhibited S. pombe growth, impaired mitochondrial membrane integrity and cellular respiration, and led to Fxn1 aggregation. Cellular iron accumulation was observed at moderate Fxn1 overexpression but was most pronounced at high levels of Fxn1. All levels of Fxn1 overexpression up-regulated oxidative stress defense and mitochondrial Fe–S cluster containing enzyme activities.

Conclusions

Despite the presence of oxidative stress and accumulated iron, activation of Fe–S cluster enzymes was common to all levels of Fxn1 overexpression; therefore, Fxn1 may regulate the efficiency of Fe–S cluster biogenesis in S. pombe.

General Significance

We provide evidence that suggests that dysregulated Fe–S cluster biogenesis is a primary effect of both frataxin overexpression and deficiency as in Friedreich's ataxia.     

Mitochondrial glutathione: Features,regulation and role in disease

Background

Mitochondria are the powerhouse of mammalian cells and the main source of reactive oxygen species (ROS) associated with oxygen consumption. In addition, they also play a strategic role in controlling the fate of cells through regulation of death pathways. Mitochondrial ROS production fulfills a signaling role through regulation of redox pathways, but also contributes to mitochondrial damage in a number of pathological states.

Scope of review

Mitochondria are exposed to the constant generation of oxidant species, and yet the organelle remains functional due to the existence of an armamentarium of antioxidant defense systems aimed to repair oxidative damage, of which mitochondrial glutathione (mGSH) is of particular relevance. Thus, the aim of the review is to cover the regulation of mGSH and its role in disease.

Major conclusions

Cumulating evidence over recent years has demonstrated the essential role for mGSH in mitochondrial physiology and disease. Despite its high concentration in the mitochondrial matrix, mitochondria lack the enzymes to synthesize GSH de novo, so that mGSH originates from cytosolic GSH via transport through specific mitochondrial carriers, which exhibit sensitivity to membrane dynamics. Depletion of mGSH sensitizes cells to stimuli leading to oxidative stress such as TNF, hypoxia or amyloid β-peptide, thereby contributing to disease pathogenesis.

General significance

Understanding the regulation of mGSH may provide novel insights to disease pathogenesis and toxicity and the opportunity to design therapeutic targets of intervention in cell death susceptibility and disease. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.     

Calorie restriction influences key metabolic enzyme activities and markers of oxidative damage in distinct mouse liver mitochondrial sub-populations

Aims

The purpose of the study was to establish if enzyme activities from key metabolic pathways and levels of markers of oxidative damage to proteins and lipids differed between distinct liver mitochondrial sub-populations, and which specific sub-populations contributed to these differences.

Main methods

Male C57BL/6J mice were fed non-purified diet for one month then separated into two groups, control and calorie-restricted (CR). The two groups were fed semi-purified diet (AIN93G), with the CR group receiving 40% less calories than controls. After two months, enzyme activities and markers of oxidative damage in mitochondria were determined.

Key findings

In all mitochondrial sub-populations, enzyme activities and markers of oxidative damage, from control and CR groups, showed a pattern of M1 > M3 > M10. Higher acyl-CoA dehydrogenase (β-oxidation) and β-hydroxybutyrate dehydrogenase (ketogenesis) activities and lower carbonyl and TBARS levels were observed in M1 and M3 fractions from CR mice. ETC enzyme activities did not show a consistent pattern. In the Krebs cycle, citrate synthase and aconitase activities decreased while succinate dehydrogenase and malate dehydrogenase activities increased in the M1 mitochondria from the CR versus control mice.

Significance

CR does not produce uniform changes in enzyme activities or markers of oxidative damage in mitochondrial sub-populations, with changes occurring primarily in the heavy mitochondrial populations. Centrifugation at 10,000 g to isolate mitochondria likely dilutes the mitochondrial populations which show the greatest response to CR. Use of lower centrifugal force (3000 g or lower) may be beneficial for some studies.     

Hypoxia controls iron metabolism and glutamate secretion in retinal pigmented epithelial cells

Background

Blood-barrier systems are essential in controlling iron levels in organs such as the brain and eye, both of which experience hypoxia in pathological conditions. While hypoxia's effects on numerous iron regulatory and storage proteins have been studied, little is known about how hypoxia affects iron metabolism. Iron also controls glutamate production and secretion; therefore the effects of hypoxia on iron metabolism and glutamate secretion were studied in polarized retinal pigmented epithelial (RPE) cells.

Methods

Primary canine RPE were cultured in Millicells to create polarized cell cultures. Iron uptake and efflux were measured in hypoxic and normoxic conditions. RPE were loaded with ⁵⁹Fe-transferrin. Glutamate concentrations in the cell conditioned media were also measured.

Results

Hypoxia induced a large increase in iron efflux from RPE in the basolateral direction. Glutamate secretion occurred mainly in the basolateral direction which is away from the retina and out of the eye in vivo. Glutamate secretion was doubled under hypoxic conditions.

Conclusions

Hypoxia is known to induce oxidative damage. The current results show that iron, a key catalyst of free radical generation, is removed from RPE under hypoxic conditions which may help protect RPE from oxidative stress. Results obtained here indicate the importance of using polarized tight junctional cells as more physiologically relevant models for blood-barrier-like systems.

General significance

While the effects of hypoxia on iron efflux and glutamate secretion may be protective for RPE cells and retina, increased glutamate secretion in the brain could cause some of the damaging neurological effects seen in stroke.     

Toxicity of depleted uranium on isolated rat kidney mitochondria

Background

Kidney is known as the most sensitive target organ for depleted uranium (DU) toxicity in comparison to other organs. Although the oxidative stress and mitochondrial damage induced by DU has been well investigated, the precise mechanism of DU-induced nephrotoxicity has not been thoroughly recognized yet.

Methods

Kidney mitochondria were obtained using differential centrifugation from Wistar rats and mitochondrial toxicity endpoints were then determined in both in vivo and in vitro uranyl acetate (UA) exposure cases.

Results

Single injection of UA (0, 0.5, 1 and 2 mg/kg, i.p.) caused a significant increase in blood urea nitrogen and creatinine levels. Isolated mitochondria from the UA-treated rat kidney showed a marked elevation in oxidative stress accompanied by mitochondrial membrane potential (MMP) collapse as compared to control group. Incubation of isolated kidney mitochondria with UA (50, 100 and 200 μM) manifested that UA can disrupt the electron transfer chain at complex II and III that leads to induction of reactive oxygen species (ROS) formation, lipid peroxidation, and glutathione oxidation. Disturbances in oxidative phosphorylation were also demonstrated through decreased ATP concentration and ATP/ADP ratio in UA-treated mitochondria. In addition, UA induced a significant damage in mitochondrial outer membrane. Moreover, MMP collapse, mitochondrial swelling and cytochrome c release were observed following the UA treatment in isolated mitochondria.

General significance

Both our in vivo and in vitro results showed that UA-induced nephrotoxicity is linked to the impairment of electron transfer chain especially at complex II and III which leads to subsequent oxidative stress.