Two pathways of iron uptake in bovine spleen apoferritin dependent on iron concentration

Iron incorporation by bovine spleen apoferritin either with ferrous ammonium sulfate in different buffers or with ferrous ammonium sulfate and phosphate was studied. Iron uptake and iron autoxidation were recorded spectrophotomerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependent iron autoxidation, with Tris showing less iron autoxidation than Hepes. An Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes was a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and of Hepes after the addition of phosphate showed a straight line. Phosphate accelerated the iron uptake rate. The iron loading kinetics of apoferritin in Hepes was dependent on apoferritin concentration. The K_m value obtained from iron uptake kinetics was 4.5 M, corresponding to the physiological iron concentration. These results demonstrate that iron loading of apoferritin was accomplished at physiological iron concentrations, which is essential for iron uptake, via two uptake pathways of dependent on iron concentration.     

Kinetic analysis of bovine spleen apoferritin and recombinant H and L chain homopolymers: Iron uptake suggests early stage H chain ferroxidase activity and second stage L chain cooperation

Ferritin utilizes ferroxidase activity to incorporate iron. Iron uptake kinetics of bovine spleen apoferritin (H: L = 1 : 1.1) were compared with those of recombinant H chain ferritin and L chain ferritin homopolymers. H chain ferritin homopolymer showed an iron uptake rate identical to bovine spleen apoferritin (0.19 and 0.21 mmol/min/micromol of protein, respectively), and both showed iron concentration-dependent uptake. In contrast, the L chain homopolymer, which lacks ferroxidase, did not incorporate iron and showed the same level of iron autoxidation in the absence of ferritin. Bovine spleen apoferritin was shown to have two iron concentration-dependent uptake pathways over a range of 0.02-0.25 mM ferrous ammonium sulfate (FAS) by an Eadie-Scatchard plot (v/[FAS] versus v), whereas the H chain ferritin homopolymer was found to have only one pathway. Of the two Km values found in bovine spleen apoferritin, the lower mean Km value was 9.0 microM, while that of the H chain homopolymer was 11.0 microM. H chain ferritin homopolymer reached a saturating iron uptake rate at 0.1 mM FAS, while bovine spleen apoferritin incorporated more iron even at 0.25 mM FAS. These results suggest that the intrinsic ferroxidase of ferritin plays a significant role in iron uptake, and the L chain cooperates with the H chain to increase iron uptake.     

Crystal structure of Streptococcus suis Dps-like peroxide resistance protein Dpr: implications for iron incorporation

The Dps-like peroxide resistance protein (Dpr) is an aerotolerance and hydrogen peroxide resistance agent found in the meningitis-associated pathogen Streptococcus suis. Dpr is believed to act by binding free intracellular iron to prevent Fenton chemistry-catalysed formation of toxic hydroxyl radicals. The crystal structure of Dpr has been determined to 1.95 A resolution. The final model has an Rcyst value of 18.5% (Rfree = 22.4%) and consists of 12 identical monomers (each of them comprising a four alpha-helix bundle) that form a hollow sphere obeying 23 symmetry. Structural features show that Dpr belongs to the Dps family of bacterial proteins. Twelve putative ferroxidase centers, each formed at the interface of neighboring monomer pairs, were identified in the Dpr structure with structural similarities to those found in other Dps family members. Dpr was crystallized in the absence of iron, hence no bound iron was found in the structure in contrast to other Dps family members. A novel metal-binding site approximately 6A from the ferroxidase centre was identified and assigned to a bound calcium ion. Two residues from the ferroxidase centre (Asp63 and Asp74) were found to be involved in calcium binding. Structural comparison with other family members revealed that Asp63 and Asp74 adopt different conformation in the Dpr structure. The structure of Dpr presented here shows potential local conformational changes that may occur during iron incorporation. A role for the metal-binding site in iron uptake is proposed.     

Unique iron binding and oxidation properties of human mitochondrial ferritin: a comparative analysis with Human H-chain ferritin

Ferritins are ubiquitous iron mineralizing and storage proteins that play an important role in iron homeostasis. Although excess iron is stored in the cytoplasm, most of the metabolically active iron is processed in the mitochondria of the cell. Little is known about how these organelles regulate iron homeostasis and toxicity. The recently discovered human mitochondrial ferritin (MtF), unlike other mammalian ferritins, is a homopolymer of 24 subunits that has a high degree of sequence homology with human H-chain ferritin (HuHF). Parallel experiments with MtF and HuHF reported here reveal striking differences in their iron oxidation and hydrolysis chemistry despite their similar diFe ferroxidase centers. In contrast to HuHF, MtF does not regenerate its ferroxidase activity after oxidation of its initial complement of Fe(II) and generally has considerably slower ferroxidation and mineralization activities as well. MtF exhibits sigmoidal kinetics of mineralization more characteristic of an L-chain than an H-chain ferritin. Site-directed mutagenesis reveals that serine 144, a residue situated near the ferroxidase center in MtF but absent from HuHF, is one player in this impairment of activity. Additionally only one-half of the 24 ferroxidase centers of MtF are functional, further contributing to its lower activity. Stopped-flow absorption spectrometry of Fe(II) oxidation by O(2) in MtF shows the formation of a transient diiron(III) mu-peroxo species (lambda(max) = 650 nm) as observed in HuHF. Also, as for HuHF, minimal hydroxyl radical is produced during the oxidative deposition of iron in MtF using O(2) as the oxidant. However, the 2Fe(II) + H(2)O(2) detoxification reaction found in HuHF does not occur in MtF. The structural differences and the physiological implications of the unique iron oxidation properties of MtF are discussed in light of these results.     

Ironing out Parkinson's disease: is therapeutic treatment with iron chelators a real possibility?

Levels of iron are increased in the brains of Parkinson's disease (PD) patients compared to age-matched controls. This has been postulated to contribute to progression of the disease via several mechanisms including exacerbation of oxidative stress, initiation of inflammatory responses and triggering of Lewy body formation. In this minireview, we examine the putative role of iron in PD and its pharmacological chelation as a prospective therapeutic for the disease.     

Differential DNA binding and protection by dimeric and dodecameric forms of the ferritin homolog Dps from Deinococcus radiodurans

Bacterial iron storage proteins such as ferritin serve as intracellular iron reserves. Members of the DNA protection during starvation (Dps) family of proteins are structurally related to ferritins, and their function is to protect the genome from iron-induced free radical damage. Some members of the Dps family bind DNA and are thought to do so only as fully assembled dodecamers. We present the cloning and characterization of a Dps homolog encoded by the radiation-resistant eubacterium Deinococcus radiodurans and show that DNA binding does not require its assembly into a dodecamer. D.radiodurans Dps-1, the product of gene DR2263, adopts a stably folded conformation, as demonstrated by circular dichroism spectroscopy, and undergoes a transition to a disordered state with a melting temperature of 69.2(+/-0.1) degrees C. While a dimeric form of Dps-1 is observed under low-salt conditions, a dodecameric assembly is highly favored at higher concentrations of salt. Both oligomeric forms of Dps-1 exhibit ferroxidase activity, and Fe(II) oxidation/mineralization is seen for dodecameric Dps-1. Notably, addition of Ca(2+) (to millimolar concentrations) to dodecameric Dps-1 can result in the reduction of bound Fe(III). Dimeric Dps-1 protects DNA from both hydroxyl radical cleavage and from DNase I-mediated cleavage; however, dodecameric Dps-1 is unable to provide efficient protection against hydroxyl radical-mediated DNA cleavage. While dodecameric Dps-1 does bind DNA, resulting in formation of large aggregates, cooperative DNA binding by dimeric Dps-1 leads to formation of protein-DNA complexes of finite stoichiometry.     

Iron induces ferritin synthesis in maize plantlets

The iron-storage protein ferritin has been purified to homogeneity from maize seeds, allowing to determine the sequence of the first 29 NH₂-terminal amino acids of its subunit and to raise specific rabbit polyclonal antibodies. Addition of 500 M Fe-EDTA/75 M Fe-citrate to hydroponic culture solutions of maize plantlets, previously starved for iron, led to a significant increase of the iron concentration of roots and leaves, albeit root iron was mainly found associated with the apoplast. Immunodetection of ferritin by western blots indicated that this iron treatment induced ferritin protein accumulation in roots and leaves over a period of 3 days. In order to investigate this induction at the ferritin mRNA level, various ferritin cDNA clones were isolated from a cDNA library prepared from poly(A)⁺ mRNA isolated from roots 48 h after iron treatment. These cDNAs were classified into two groups called FM1 and FM2. Upstream of the sequence encoding the mature ferritin subunit, both of these cDNAs contained an in-frame coding sequence with the characteristics of a transit peptide for plastid targeting. Two members of the FM1 subfamily, both partial at their 5 extremity, were characterized. They are identical, except in their 3 untranslated region: FM1A extends 162 nucleotides beyond the 3 terminus of FM1B. These two mRNAs could arise from the use of two different polyadenylation signals. FM2 is 96% identical to FM1 and contains 45 nucleotides of 5 untranslated region. Northern analyses of root and leaf RNAs, at different times after iron treatment, revealed ferritin mRNA accumulation in response to iron. Ferritin mRNA accumulation was transient and particularly abundant in leaves, reaching a maximum at 24 h. The level of ferritin mRNA in roots was affected to a lesser extent than in leaves.     

外源一氧化碳对铁胁迫下的离体水稻叶片氧化伤害的缓解作用

不同饱和度的外源CO水溶液预处理离体水稻叶片,可以浓度依赖性地缓解铁胁迫引起的硫代巴比妥酸反应物质（TBARS）含量的上升,并诱导铁蛋白基因表达。此外,50%饱和度的CO水溶液还能不同程度地逆转由于柠檬酸铁胁迫引起的叶中抗坏血酸过氧化物酶（APX）和超氧化物歧化酶（SOD）活性的上升。     

A Diatom Ferritin Optimized for Iron Oxidation but Not Iron Storage

Ferritin from the marine pennate diatom Pseudo-nitzschia multiseries (PmFTN) plays a key role in sustaining growth in iron-limited ocean environments. The di-iron catalytic ferroxidase center of PmFTN (sites A and B) has a nearby third iron site (site C) in an arrangement typically observed in prokaryotic ferritins. Here we demonstrate that Glu-44, a site C ligand, and Glu-130, a residue that bridges iron bound at sites B and C, limit the rate of post-oxidation reorganization of iron coordination and the rate at which Fe³⁺ exits the ferroxidase center for storage within the mineral core. The latter, in particular, severely limits the overall rate of iron mineralization. Thus, the diatom ferritin is optimized for initial Fe²⁺ oxidation but not for mineralization, pointing to a role for this protein in buffering iron availability and facilitating iron-sparing rather than only long-term iron storage.     

Changes in skeletal muscle iron metabolism outpace amyotrophic lateral sclerosis onset in transgenic rats bearing the G93A hmSOD1 gene mutation

Abstract

Objective. Recently, iron and the adaptor protein “p66Shc” have been shown to play an important role in the development of amyotrophic lateral sclerosis (ALS) in rats. We hypothesized that changes in muscle p66Shc activity and iron metabolism would appear before visible symptoms of the disease occurred. Methods. In the present study, we used transgenic rats bearing the G93A hmSOD1 gene mutation and their non-transgenic littermates to test this hypothesis. We examined muscle p66Shc phosphorylation and iron metabolism in relation to oxidative stress in animals at three disease stages: asymptomatic (ALS I), disease onset (ALS II), and end-stage disease (ALS III). Results. Significant changes in iron metabolism and markers of lipid and protein oxidation were detected in ALS I animals, which manifested as decreased levels of ferritin H and ferroportin 1 (Fpn1) and increased levels of ferritin L levels. Muscles of ALS I rats possessed increased levels of p66Shc phosphorylated at Ser³⁶ compared with muscles of control rats. During disease progression, level of ferritin H significantly increased and was accompanied by iron accumulation. Conclusions. This study showed that multiple mechanisms may underlie iron accumulation in muscles of ALS transgenic rats, which include changes in blood hepcidin and muscle Fpn1 and increased level of muscle ferritin H. These data suggest that impaired iron metabolism is not a result of changes in motor activity.     

微生物铁蛋白的研究进展

铁蛋白作为一种重要的铁储存蛋白,在不同的微生物体中普遍存在.通过对典型的微生物铁蛋白分子(FTN)的结构及其功能的归纳分析发现,铁蛋白依赖其独特的结构特点,在铁的补充、转运、氧化、成核和储存中扮演着重要作用,也对生物体内的多种生物化学反应影响显著.同时借助基因工程技术对铁蛋白进行相应的分子改造,增加了其作为纳米载体的应...     

胎盘铁转运蛋白Zyklopen研究进展

Zyklopen（ZP）作为铜蓝蛋白的同系物,是近年来发现的铁转运蛋白.ZP具有一个位于羧基末端的跨膜结构域,在胎盘大量表达,也分布于脑、肾、视网膜、乳腺、睾丸等组织,但目前还不清楚ZP在这些组织中的功能.ZP具有亚铁氧化酶的活性,细胞内缺铜会引起ZP蛋白表达减少.在胎盘中,ZP可能通过将二价铁离子氧化为三价铁离子,帮助三价铁与胎儿循环系统中的转铁蛋白相结合,从而参与铁从母体到胎儿的转运过程.     

Self-assembly Is Prerequisite for Catalysis of Fe(II) Oxidation by Catalytically Active Subunits of Ferritin

Fe(III) storage by ferritin is an essential process of the iron homeostasis machinery. It begins by translocation of Fe(II) from outside the hollow spherical shape structure of the protein, which is formed as the result of self-assembly of 24 subunits, to a di-iron binding site, the ferroxidase center, buried in the middle of each active subunit. The pathway of Fe(II) to the ferroxidase center has remained elusive, and the importance of self-assembly for the functioning of the ferroxidase center has not been investigated. Here we report spectroscopic and metal ion binding studies with a mutant of ferritin from Pyrococcus furiosus (PfFtn) in which self-assembly was abolished by a single amino acid substitution. We show that in this mutant metal ion binding to the ferroxidase center and Fe(II) oxidation at this site was obliterated. However, metal ion binding to a conserved third site (site C), which is located in the inner surface of each subunit in the vicinity of the ferroxidase center and is believed to be the path for Fe(II) to the ferroxidase center, was not disrupted. These results are the basis of a new model for Fe(II) translocation to the ferroxidase center: self-assembly creates channels that guide the Fe(II) ions toward the ferroxidase center directly through the protein shell and not via the internal cavity and site C. The results may be of significance for understanding the molecular basis of ferritin-related disorders such as neuroferritinopathy in which the 24-meric structure with 432 symmetry is distorted.