Bleomycin-Detectable Iron in Brain Tissue

The normal brain contains regions with high concentrations of iron, part of which appears to be in a low molecular mass chelatable form. Iron complexes with a molecular mass of below 10,000, were measured in ultrafiltrates of homogenized gerbil brains using the bleomycin assay, and were found to average 20.5 ± 3.5 μM (n = 8). As expected, no bleomycin detectable iron was found in the plasma of these animals.

No obvious difference in the tissue levels of bleomycin-detectable iron was recorded following ischaemia and reperfusion. This is probably due to the already abundant presence of iron in the brain and the likely release of iron from protected sites due to structural damage inherent in the preparative procedures used.     

氧化还原对铁结合的大肠杆菌拓扑异构酶Ⅰ活性的调控

大肠杆菌拓扑异构酶 I（E. coli TopA）属于 I 型拓扑异构酶,在DNA复制、转录、重组和基因表达调控等过程中发挥关键作用。E. coli TopA 不仅能结合锌,还可以结合铁。细胞内过量铁可与锌竞争,通过与锌指结构域结合减弱其 DNA 结合能力和改变蛋白质空间构象,从而抑制TopA拓扑异构酶活性。然而,铁结合形式TopA的氧化还原特性以及氧化还原条件对其活性的影响仍不清楚。本研究通过紫外分光光谱和体外DNA拓扑异构酶活性分析,发现体外纯化得到的铁结合形式的 TopA 呈氧化状态,能够被二硫苏糖醇和连二亚硫酸钠还原,原本氧化状态下无活性的TopA在还原条件下,可恢复其拓扑异构酶活性。当还原剂被去除后,铁结合的TopA在空气中能够重新被氧化,且其活性重新受到抑制。这说明,氧化还原条件对铁结合的 TopA 功能具有可逆调节作用。通过金属 蛋白体外结合实验进一步发现,无金属结合的TopA蛋白(apo-TopA)在无氧条件下,与 Fe²⁺ 和 Fe³⁺ 均能结合,但与Fe²⁺ 结合能力较弱,并且TopA结合的Fe³⁺ 被还原成Fe²⁺ 后,结合力显著下降,能够被铁螯合指示剂菲咯嗪快速捕获。此外,蛋白质内源性荧光光谱分析实验表明,铁结合的TopA在氧化还原的不同状态时,其在330 nm左右的荧光值有显著差异。这提示,氧化还原条件可能通过影响铁离子与TopA的结合状态,引起蛋白质空间构象改变,从而对TopA的拓扑异构酶活性进行调节。此研究表明,铁结合TopA的拓扑异构酶活性会受到细胞内氧化还原信号的可逆调控,也提示I型拓扑异构酶可能是细胞铁超载通过氧化损伤引起细胞功能障碍（或铁死亡）的靶点之一。     

Structural basis of the zinc- and terbium-mediated inhibition of ferroxidase activity in Dps ferritin-like proteins

Streptococcus suis Dpr is an iron-binding protein involved in oxidative stress resistance. It belongs to the bacterial Dps protein family whose members form dodecameric assemblies. Previous studies have shown that zinc and terbium inhibit iron incorporation in Listeria innocua Dps protein. In order to gain structural insights into the inhibitory effect of zinc and terbium, the crystal structures of Streptococcus suis Dpr complexes with these ions were determined at 1.8 A and 2.1 A, respectively. Both ions were found to bind at the ferroxidase center and in the same location as iron. In addition, a novel zinc-binding site formed by His40 and His44 was identified. Both His residues were found to be present within all known Streptococcus suis Dpr variants and in Streptococcus pneumoniae, Streptococcus gordonii, and Streptococcus sanguinis Dpr proteins. Amino acid sequence alignment of Dpr with other Dps family members revealed that His44 is highly conserved, in contrast to His40. The inhibitory effect of zinc and terbium on iron oxidation in Dpr was studied in vitro, and it was found that both ions at concentrations >0.2 mM almost completely abolish iron binding. These results provide a structural basis for the inhibitory effect of zinc and terbium in the Dps family of proteins, and suggest a potential role of the Dps proteins in zinc detoxification mechanisms involving the second zinc-binding site.     

铁核结构对马脾铁蛋白释放铁动力学的影响

建立Ｈ^% 参与马脾铁蛋白释放铁的动力方程,Ｈ^ 以１／２级反应方式参与铁蛋白释放铁核表层的铁。在酸性介质（ＰＨ６．５）中,铁蛋白释放铁的总平均速率（３３２Ｆe^3 /HSF.min)比在碱性介质（Ｐ8Ｈ８．０）中放铁的总平均速率（７３Ｆe^3 /HSF.min)高４．６倍,铁蛋白的铁核结构和外加的磷酸盐均能影响该蛋白释放的速率,但并不改变其反应级数。     

Combination of Iron Overload Plus Ethanol and Ischemia Alone Give Rise to the Same Endogenous Free Iron Pool

Iron overload aggravates tissue damage caused by ischemia and ethanol intoxication. The underlying mechanisms of this phenomenon are not yet clear. To clarify these mechanisms we followed free iron (“loosely” bound redox-active iron) concentration in livers from rats subjected to experimental iron overload, acute ethanol intoxication, and ex vivo warm ischemia. The levels of free iron in non-homogenized liver tissues, liver homogenates, and hepatocyte cultures were analyzed by means of EPR spectroscopy. Ischemia gradually increased the levels of endogenous free iron in liver tissues and in liver homogenates. The increase was accompanied by the accumulation of lipid peroxidation products. Iron overload alone, known to increase significantly the total tissue iron, did not affect either free iron levels or lipid peroxidation. Homogenization of iron-loaded livers, however, resulted in the release of a significant portion of free iron from endogenous depositories. Acute ethanol intoxication increased free iron levels in liver tissue and diminished the portion of free iron releasing during homogenization. Similarly to liver tissue, the primary hepatocyte culture loaded with iron in vitro released significantly more free iron during homogenization compared to non iron-loaded hepatocyte culture. Analyzing three possible sources of free iron release under these experimental conditions in liver cells, namely ferritin, intracellular transferrin-receptor complex and heme oxygenase, we suggest that redox active free iron is released from ferritin under ischemic conditions whereas ethanol and homogenization facilitate the release of iron from endosomes containing transferrin-receptor complexes.     

Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides

Hydrogen peroxide and organic hydroperoxides react with haemoglobin to release iron which can be complexed to apotransferrin, bleomycin and desferrioxamine. This released iron promotes deoxyribose degradation by a Fenton reaction, DNA degradation in the presence of bleomycin and stimulates lipid peroxidation. It is likely that iron released from haemoglobin is the true generator of hydroxyl radicals in the Fenton reaction.     

Iron inhibits Escherichia coli topoisomerase I activity by targeting the first two zinc‐binding sites in the C‐terminal domain

Escherichia coli DNA topoisomerase I (TopA) contains a 67 kDa N‐terminal catalytic domain and a 30 kDa C‐terminal zinc‐binding region (ZD domain) which has three adjacent tetra‐cysteine zinc‐binding motifs. Previous studies have shown that E. coli TopA can bind both iron and zinc, and that iron binding in TopA results in failure to unwind the negatively supercoiled DNA. Here, we report that each E. coli TopA monomer binds one atom of iron via the first two zinc‐binding motifs in ZD domain and both the first and second zinc‐binding motifs are required for iron binding in TopA. The site‐directed mutagenesis studies further reveal that while the mutation of the third zinc‐binding motif has very little effect on TopA's activity, mutation of the first two zinc‐binding motifs in TopA greatly diminishes the topoisomerase activity in vitro and in vivo, indicating that the first two zinc‐binding motifs in TopA are crucial for its function. The DNA‐binding activity assay and intrinsic tryptophan fluorescence measurements show that iron binding in TopA may decrease the single‐stranded (ss) DNA‐binding activity of ZD domain and also change the protein structure of TopA, which subsequently modulate topoisomerase activity.     

Binding of mineral elements to locust bean gum influences availability in vitro

Experiments were conducted to evaluate the extent to which element binding of locust bean gum (LBG) affects the availability of calcium, iron, and zinc in the gut. Infant formula was supplemented with increasing amounts of LBG and subjected to an intraluminal digestion procedure. Element binding was measured by eliminating the complexes by twofold centrifugation. Availability of the elements was determined using a validated continuous-flow dialysis technique. Elemental content of the samples, supernatants, and dialysates was analyzed with validated atomic absorption spectrometry. LBG provided small amounts of intrinsic calcium (1.13 ± 0.02 mg/g) and trace amounts of iron (0.02 ± 0.00 mg/g) and zinc (0.01 ± 0.00 mg/g), which were strongly bound to the LBG molecule (respectively: 76.6 ± 3.3%, 83.4 ± 1.2%, 96.7 ± 6.6%). Correlation analysis, between percent element bound by LBG after centrifugation and percent trapped after dialysis, yielded significant correlation only for the data of zinc (r=0.93). For calcium and iron, no correlation could be demonstrated; however, for iron a similar trend was observed. These findings suggest that element binding of LBG has a major influence on the availability of zinc and maybe of iron. For calcium, other factors might also be involved, affecting availability.     

Iron uptake by Caco-2 cells following in vitro digestion: Effects of heat treatments of pork meat and pH of the digests

The present in vitro studies report on iron uptake by Caco-2 cells from pepsin and pepsin + pancreatin-digested pork meat proteins at pH values between 4.6 and 7 mimicking conditions in the duodenum and the proximal jejunum, respectively. Heat treatment of the pork meat resulted in increased iron uptake from pepsin-digested samples to Caco-2 cells at pH 4.6. The major enhancing effects on iron uptake by Caco-2 cells were observed after pepsin digestion in the pH range 4.6–6.0, whereas the pepsin + pancreatin-digested samples resulted in negligible iron uptake in Caco-2 cells at pH 7. Thus, the results emphasize the importance of separating pepsin-digested and pepsin + pancreatin-digested proteins during in vitro studies on iron availability. Furthermore, the present results showed the pH dependency of iron uptake anticipated. The enhancing effect of ascorbic acid was verified by increased iron uptake from pepsin-digested pork meat samples at pH 4.6, while no effect of ascorbic acid was observed at pH 7 in pepsin + pancreatin-digested samples.     

Effect of lipids and organic solvents on the enzymic formation of zinc protoporphyrin and haem

1. Differences observed in earlier work between the enzymic chelation with protoporphyrin of Zn(2+) and Fe(2+) ions respectively have now been explained as being caused by the presence of peroxides in the ether used in the enzyme assay. The inhibitory effect of peroxides is established by the reducing agent, which is present in the assay for chelation of iron but not in that for zinc. There are now no reasons for the belief that two different enzymes catalyse formation of complexes with zinc and iron respectively. 2. Removal of lipid from both chromatophores and mitochondria markedly reduced chelatase activity. Activity could be partially restored by the addition of lipid fractions. Phosphatidic acid, but not phosphatidylcholine or phosphatidylethanolamine, actively stimulated the formation of zinc protoporphyrin and haem by chromatophores and mitochondrial preparations. 3. Lipid-containing extracts of chromatophores, and fractions thereof obtained by silicic acid chromatography, partially restored chelatase activity of Tween extracts of mitochondria. Thus, although both enzymes are considered to be lipoproteins, the identity of the lipids concerned is still uncertain. 4. A great number of organic solvents such as esters, ethers, ketones and, to a lesser extent, alcohols, stimulate enzymic chelation of both metals with protoporphyrin. A number of explanations for these findings are considered and it is suggested that organic solvents interact in some way with the enzyme lipoprotein, changing either its conformation or allowing closer contact between the enzyme and its substrates.     

Crystal Structure of Bfr A from Mycobacterium tuberculosis: Incorporation of Selenomethionine Results in Cleavage and Demetallation of Haem

Emergence of tuberculosis as a global health threat has necessitated an urgent search for new antitubercular drugs entailing determination of 3-dimensional structures of a large number of mycobacterial proteins for structure-based drug design. The essential requirement of ferritins/bacterioferritins (proteins involved in iron storage and homeostasis) for the survival of several prokaryotic pathogens makes these proteins very attractive targets for structure determination and inhibitor design. Bacterioferritins (Bfrs) differ from ferritins in that they have additional noncovalently bound haem groups. The physiological role of haem in Bfrs is not very clear but studies indicate that the haem group is involved in mediating release of iron from Bfr by facilitating reduction of the iron core. To further enhance our understanding, we have determined the crystal structure of the selenomethionyl analog of bacterioferritin A (SeMet-BfrA) from Mycobacterium tuberculosis (Mtb). Unexpectedly, electron density observed in the crystals of SeMet-BfrA analogous to haem location in bacterioferritins, shows a demetallated and degraded product of haem. This unanticipated observation is a consequence of the altered spatial electronic environment around the axial ligands of haem (in lieu of Met52 modification to SeMet52). Furthermore, the structure of Mtb SeMet-BfrA displays a possible lost protein interaction with haem propionates due to formation of a salt bridge between Arg53-Glu57, which appears to be unique to Mtb BfrA, resulting in slight modulation of haem binding pocket in this organism. The crystal structure of Mtb SeMet-BfrA provides novel leads to physiological function of haem in Bfrs. If validated as a drug target, it may also serve as a scaffold for designing specific inhibitors. In addition, this study provides evidence against the general belief that a selenium derivative of a protein represents its true physiological native structure.     

Iron autoxidation and free radical generation: effects of buffers, ligands, and chelators.

The pH of the solution along with chelation and consequently coordination of iron regulate its reactivity. In this study we confirmed that, in general, the rate of Fe(II) autoxidation increases as the pH of the solution is increased, but chelators that provide oxygen ligands for the iron can override the affect of pH. Additionally, the stoichiometry of the Fe(II) autoxidation reaction varied from 2:1 to 4:1, dependent upon the rate of Fe(II) autoxidation, which is dependent upon the chelator. No partially reduced oxygen species were detected during the autoxidation of Fe(II) by ESR using DMPO as the spin trap. However, upon the addition of ethanol to the assay, the DMPO:hydroxyethyl radical adduct was detected. Additionally, the hydroxylation of terephthalic acid by various iron-chelator complexes during the autoxidation of Fe(II) was assessed by fluorometric techniques. The oxidant formed during the autoxidation of EDTA:Fe(II) was shown to have different reactivity than the hydroxyl radical, suggesting that some type of hypervalent iron complex was formed. Ferrous iron was shown to be able to directly reduce some quinones without the reduction of oxygen. In conclusion, this study demonstrates the complexity of iron chemistry, especially the chelation of iron and its subsequent reactivity.     

Fe-haem bound to Escherichia coli bacterioferritin accelerates iron core formation by an electron transfer mechanism

BFR (bacterioferritin) is an iron storage and detoxification protein that differs from other ferritins by its ability to bind haem cofactors. Haem bound to BFR is believed to be involved in iron release and was previously thought not to play a role in iron core formation. Investigation of the effect of bound haem on formation of the iron core has been enabled in the present work by development of a method for reconstitution of BFR from Escherichia coli with exogenously added haem at elevated temperature in the presence of a relatively high concentration of sodium chloride. Kinetic analysis of iron oxidation by E. coli BFR preparations containing various amounts of haem revealed that haem bound to BFR decreases the rate of iron oxidation at the dinuclear iron ferroxidase sites but increases the rate of iron core formation. Similar kinetic analysis of BFR reconstituted with cobalt-haem revealed that this haem derivative has no influence on the rate of iron core formation. These observations argue that haem bound to E. coli BFR accelerates iron core formation by an electron-transfer-based mechanism.     

Iron release analyses from ferritin by visible light irradiation

We investigated the iron release from ferritin by irradiation from a white fluorescent light in the absence or presence of ADP. Irradiation of a ferritin solution at 17,000 lx in the absence of ADP slightly induces iron release from ferritin but only at acidic pH conditions (pH 5.0 or pH 6.0). Irradiation in the presence of ADP markedly enhances iron release from ferritin under the same conditions. In the absence of irradiation, the iron release from ferritin was low even in the presence of ADP. The induction of the iron release by irradiation in the presence of ADP was also affected by various factors such as irradiation dose and acidity, but not temperature (4-47°C), oxygen concentration, or free radical generations during the irradiation. The iron release during the irradiation ceased to increase by turning off the light and was found to increase again after additional irradiation. These results suggest that visible light directly induces iron release from ferritin via the photoreduction of iron stored inside ferritin.     

Non-transferrin bound iron measurement is influenced by chelator concentration

Release of non-protein bound iron plays an important role in the toxicity inflicted by chemotherapy in cancer patients. Since large variations have been described for different methods measuring non-transferrin bound iron (NTBI), we aimed to obtain more accurate values. After binding to the chelator nitrilotriacetic acid disodium salt (NTA) and ultrafiltration, the NTBI can be measured spectrophotometrically by the addition of thioglycolic acid (TGA) and baptophenanthroline disulfonic acid (BPT). Results demonstrated that NTBI values increased with NTA concentration. In samples incubated with 80 mM NTA, >5-fold higher NTBI values were found compared to using 10 mM NTA. Optimal concentration of NTA was established by additions of iron to serum with known latent iron-binding capacity (LIBC). Iron addition curves showed that NTBI could be measured starting from the LIBC of the serum with optimal yield after incubation with 4 mM NTA in 5 mM Tris-HCl pH 6.5, with 3 mM TGA and 6.2 mM BPT for the colour reaction. The results showed excellent correlation with 195 samples measured also by HPLC. For the spectrophotometric method, significantly higher NTBI values were measured in patient samples with maximal iron saturation compared to patients with lower iron saturation.     

Ascorbate-mediated Iron Release from Ferritin in the Presence of Alloxan

Release of iron from ferritin requires reduction of ferric to ferrous iron. The iron can participate in the diabetogenic action of alloxan. We investigated the ability of ascorbate to catalyze the release of iron from ferritin in the presence of alloxan. Incubation of ferritin with ascorbate alone elicited iron release (33 nmol/10 min) and the generation of ascorbate free radical, suggesting a direct role for ascorbate in iron reduction. Iron release by ascorbate significantly increased in the presence of alloxan, but alloxan alone was unable to release measurable amounts of iron from ferritin. Superoxide dismutase significantly inhibited ascorbate-mediated iron release in the presence of alloxan, whereas catalase did not. The amount of alloxan radical (A·⁻) generated in reaction systems containing both ascorbate and alloxan decreased significantly upon addition of ferritin, suggesting that A·⁻ is directly involved in iron reduction. Although release of iron from ferritin and generation of A·⁻ were also observed in reactions containing GSH and alloxan, the amount of iron released in these reactions was not totally dependent on the amount of A·⁻ present, suggesting that other reductants in addition to A·⁻ (such as dialuric acid) may be involved in iron release mediated by GSH and alloxan. These results suggest that A·⁻ is the main reductant involved in ascorbate-mediated iron release from ferritin in the presence of alloxan and that both dialuric acid and A·⁻ contribute to GSH/alloxan-mediated iron release.     

Involvement of Vulnibactin and Exocellular Protease in Utilization of Transferrin- and Lactoferrin-Bound Iron by Vibrio vulnificus

In vitro growth experiments were conducted to evaluate the ability of vulnibactin, a siderophore produced by Vibrio vulnificus, to sequester transferrin- or lactoferrin-bound iron for growth. Comparative studies with the strain producing vulnibactin and its exocellular protease-deficient mutant revealed the involvement of the protease in addition to vulnibactin in effective utilization of iron ion (Fe³⁺) bound to transferrin and lactoferrin. It appears that the protease causes cleavage of these proteins, thereby making bound iron more accessible to vulnibactin.     

Effect of pH and oxalic acid on the reduction of Fe3+ by a biomimetic chelator and on Fe3+ desorption/adsorption onto wood: Implications for brown-rot decay

Fenton reaction is thought to play an important role in wood degradation by brown-rot fungi. In this context, the effect of oxalic acid and pH on iron reduction by a biomimetic fungal chelator and on the adsorption/desorption of iron to/from wood was investigated. The results presented in this work indicate that at pH 2.0 and 4.5 and in the presence of oxalic acid, the phenolate chelator 2,3-dihydroxybenzoic acid (2,3-DHBA) is capable of reducing ferric iron only when the iron is complexed with oxalate to form Fe³⁺-mono-oxalate (Fe(C₂O₄)⁺). Within the pH range tested in this work, this complex formation occurs when the oxalate:Fe³⁺ molar ratio is less than 20 (pH 2.0) or less than 10 (pH 4.5). When aqueous ferric iron was passed through a column packed with milled red spruce (Picea rubens) wood equilibrated at pH 2.0 and 4.5, it was observed that ferric iron binds to wood at pH 4.5 but not at pH 2.0, and the bound iron could then be released by application of oxalic acid at pH 4.5. The release of bound iron was dependent on the amount of oxalic acid applied in the column. When the amount of oxalate was at least 20-fold greater than the amount of iron bound to the wood, all bound iron was released. When Fe–oxalate complexes were applied to the milled wood column equilibrated in the pH range of 2–4.5, iron from Fe–oxalate complexes was bound to the wood only when the pH was 3.6 or higher and the oxalate:Fe³⁺ molar ratio was less than 10. When 2,3-DHBA was evaluated for its ability to release iron bound to the milled wood, it was found that 2,3-DHBA possessed a greater affinity for ferric iron than the wood as 2,3-DHBA was capable of releasing the ferric iron bound to the wood in the pH range 3.6–5.5. These results further the understanding of the mechanisms employed by brown-rot fungi in wood biodegradation processes.     

Iron acquisition systems in the pathogenic Neisseria

Pathogenic neisseriae have a repertoire of high-affinity iron uptake systems to facilitate acquisition of this essential element in the human host. They possess surface receptor proteins that directly bind the extracellular host iron-binding proteins transferrin and lactoferrin. Alternatively, they have siderophore receptors capable of scavenging iron when exogenous siderophores are present. Released intracellular haem iron present in the form of haemoglobin, haemoglobin-haptoglobin or free haem can be used directly as a source of iron for growth through direct binding by specific surface receptors. Although these receptors may vary in complexity and composition, the key protein involved in the transport of iron (as iron, haem or iron-siderophore) across the outer membrane is a TonB-dependent receptor with an overall structure presumably similar to that determined recently for Escherichia coli FhuA or FepA. The receptors are potentially ideal vaccine targets in view of their critical role in survival in the host. Preliminary pilot studies indicate that transferrin receptor-based vaccines may be protective in humans.     

Iron binding to Azotobacter salinestris melanin,iron mobilization and uptake mediated by siderophores

Iron-sufficient Azotobacter salinestris cells bound large amounts of ⁵⁵Fe to cell-associated catechol melanin in an energy-independent manner. Iron was mobilized from the cell surface by citric acid and transported into the cell in a process that was inhibited by azide, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), KCl or RbCl, the latter two known to inhibit Na⁺-dependent activities in A. salinestris. Iron-limited cells produced a hydroxamate compound (HDX) which promoted ⁵⁵Fe-uptake into iron-limited cells in a two step process. Initial uptake was inhibited by azide or CCCP, but not by KCl, while subsequent uptake was blocked by all inhibitors. Citric acid also mediated energy-dependent ⁵⁵Fe-uptake in iron-limited cells, but initial iron-uptake was less sensitive to CCCP than HDX-mediated iron-uptake. The results show that melanin serves as an iron trap, probably to protect the cells from oxidative damage mediated by H₂O₂ and the Fenton reaction. A model for HDX siderophore-mediated iron-uptake is proposed which requires energy to concentrate iron in the periplasm and H⁺/Na⁺-dependent events to bring iron into the cell.